Antigen binding molecules that bind EGFR, vectors encoding same, and uses thereof

ABSTRACT

The present invention relates to antigen binding molecules (ABMs). In particular embodiments, the present invention relates to recombinant monoclonal antibodies, including chimeric, primatized or humanized antibodies specific for human EGFR. In addition, the present invention relates to nucleic acid molecules encoding such ABMs, and vectors and host cells comprising such nucleic acid molecules. The invention further relates to methods for producing the ABMs of the invention, and to methods of using these ABMs in treatment of disease. In addition, the present invention relates to ABMs with modified glycosylation having improved therapeutic properties, including antibodies with increased Fc receptor binding and increased effector function.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antigen binding molecules (ABMs). Inparticular embodiments, the present invention relates to recombinantmonoclonal antibodies, including chimeric, primatized or humanizedantibodies specific for human epidermal growth factor receptor (EGFR).In addition, the present invention relates to nucleic acid moleculesencoding such ABMs, and vectors and host cells comprising such nucleicacid molecules. The invention further relates to methods for producingthe ABMs of the invention, and to methods of using these ABMs intreatment of disease. In addition, the present invention relates to ABMswith modified glycosylation having improved therapeutic properties,including antibodies with increased Fc receptor binding and increasedeffector function.

2. Background Art

EGFR and Anti-EGFR Antibodies

Human epidermal growth factor receptor (also known as HER-1 or Erb-B1,and referred to herein as “EGFR”) is a 170 kDa transmembrane receptorencoded by the c-erbB protooncogene, and exhibits intrinsic tyrosinekinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996);Herbst and Shin, Cancer 94:1593-1611 (2002)). SwissProt database entryP00533 provides the sequence of EGFR. There are also isoforms andvariants of EGFR (e.g., alternative RNA transcripts, truncated versions,polymorphisms, etc.) including but not limited to those identified bySwissprot database entry numbers P00533-1, P00533-2, P00533-3, andP00533-4. EGFR is known to bind ligands including epidermal growthfactor (EGF), transforming growth factor-α (TGf-α), amphiregulin,heparin-binding EGF (hb-EGF), betacellulin, and epiregulin (Herbst andShin, Cancer 94:1593-1611 (2002); Mendelsohn and Baselga, Oncogene19:6550-6565 (2000)). EGFR regulates numerous cellular processes viatyrosine-kinase mediated signal transduction pathways, including, butnot limited to, activation of signal transduction pathways that controlcell proliferation, differentiation, cell survival, apoptosis,angiogenesis, mitogenesis, and metastasis (Atalay et al., Ann. Oncology14:1346-1363 (2003); Tsao and Herbst, Signal 4:4-9 (2003); Herbst andShin, Cancer 94:1593-1611 (2002); Modjtahedi et al., Br. J Cancer73:228-235 (1996)).

Overexpression of EGFR has been reported in numerous human malignantconditions, including cancers of the bladder, brain, head and neck,pancreas, lung, breast, ovary, colon, prostate, and kidney. (Atalay etal., Ann. Oncology 14:1346-1363 (2003); Herbst and Shin, Cancer94:1593-1611 (2002) Modjtahedi et al., Br. J Cancer 73:228-235 (1996)).In many of these conditions, the overexpression of EGFR correlates or isassociated with poor prognosis of the patients. (Herbst and Shin, Cancer94:1593-1611 (2002) Modjtahedi et al., Br. J Cancer 73:228-235 (1996)).EGFR is also expressed in the cells of normal tissues, particularly theepithelial tissues of the skin, liver, and gastrointestinal tract,although at generally lower levels than in malignant cells (Herbst andShin, Cancer 94:1593-1611 (2002)).

Unconjugated monoclonal antibodies (mAbs) can be usefull medicines forthe treatment of cancer, as demonstrated by the U.S. Food and DrugAdministration's approval of Trastuzumab (Herceptin™; Genentech Inc,)for the treatment of advanced breast cancer (Grillo-Lopez, A.-J., etal., Semin. Oncol. 26:66-73 (1999); Goldenberg, M. M., Clin. Ther.21:309-18 (1999)), Rituximab (Rituxan™; IDEC Pharmaceuticals, San Diego,Calif., and Genentech Inc., San Francisco, Calif.), for the treatment ofCD20 positive B-cell, low-grade or follicular Non-Hodgkin's lymphoma,Gemtuzumab (Mylotarg™, Celltech/Wyeth-Ayerst) for the treatment ofrelapsed acute myeloid leukemia, and Alemtuzumab (CAMPATH™, MilleniumPharmaceuticals/Schering AG) for the treatment of B cell chroniclymphocytic leukemia. The success of these products relies not only ontheir efficacy but also on their outstanding safety profiles(Grillo-Lopez, A. J., et al., Semin. Oncol. 26:66-73 (1999); Goldenberg,M. M., Clin. Ther. 21:309-18 (1999)). In spite of the achievements ofthese drugs, there is currently a large interest in obtaining higherspecific antibody activity than what is typically afforded byunconjugated mAb therapy.

The results of a number of studies suggest that Fc-receptor-dependentmechanisms contribute substantially to the action of cytotoxicantibodies against tumors and indicate that an optimal antibody againsttumors would bind preferentially to activation Fc receptors andminimally to the inhibitory partner FcγRIIB. (Clynes, R. A., et al.,Nature Medicine 6(4):443-446 (2000); Kalergis, A. M., and Ravetch, J.V., J. Exp. Med. 195(12):1653-1659 (June 2002). For example, the resultsof at least one study suggest that polymorphism in the FcγRIIIareceptor, in particular, is strongly associated with the efficacy ofantibody therapy. (Cartron, G., et al., Blood 99(3):754-757 (February2002)). That study showed that patients homozygous for FcγRIIIa have abetter response to Rituximab than heterozygous patients. The authorsconcluded that the superior response was due to better in vivo bindingof the antibody to FcγRIIIa, which resulted in better ADCC activityagainst lymphoma cells. (Cartron, G., et al., Blood 99(3):754-757(February 2002)).

Various strategies to target EGFR and block EGFR signaling pathways havebeen reported. Small-molecule tyrosine kinase inhibitors like gefitinib,erlotinib, and CI-1033 block autophosphorylation of EGFR in theintracellular tyrosine kinase region, thereby inhibiting downstreamsignaling events (Tsao and Herbst, Signal 4: 4-9 (2003)). Monoclonalantibodies, on the other hand, target the extracellular portion of EGFR,which results in blocking ligand binding and thereby inhibits downstreamevents such as cell proliferation (Tsao and Herbst, Signal 4: 4-9(2003)).

Several murine monoclonal antibodies have been generated which achievesuch a block in vitro and which have been evaluated for their ability toaffect tumor growth in mouse xenograft models (Masui, et al., CancerRes. 46:5592-5598 (1986); Masui, et al., Cancer Res. 44:1002-1007(1984); Goldstein, et al., Clin. Cancer Res. 1: 1311-1318 (1995)). Forexample, EMD 55900 (EMD Pharmaceuticals) is a murine anti-EGFRmonoclonal antibody that was raised against human epidermoid carcinomacell line A431 and was tested in clinical studies of patients withadvanced squamous cell carcinoma of the larynx or hypopharynx (Bier etal., Eur. Arch. Otohinolaryngol. 252:433-9 (1995)). In addition, the ratmonoclonal antibodies ICR16, ICR62, and ICR80, which bind theextracellular domain of EGFR, have been shown to be effective atinhibiting the binding of EGF and TGF-α the receptor. (Modjtahedi etal., Int. J. Cancer 75:310-316 (1998)). The murine monoclonal antibody425 is another MAb that was raised against the human A431 carcinoma cellline and was found to bind to a polypeptide epitope on the externaldomain of the human epidermal growth factor receptor. (Murthy et al.,Arch. Biochem. Biophys. 252(2):549-560 (1987). A potential problem withthe use of murine antibodies in therapeutic treatments is that non-humanmonoclonal antibodies can be recognized by the human host as a foreignprotein; therefore, repeated injections of such foreign antibodies canlead to the induction of immune responses leading to harmfulhypersensitivity reactions. For murine-based monoclonal antibodies, thisis often referred to as a Human Anti-Mouse Antibody response, or “HAMA”response, or a Human Anti-Rat Antibody, or “HARA” response.Additionally, these “foreign” antibodies can be attacked by the immunesystem of the host such that they are, in effect, neutralized beforethey reach their target site. Furthermore, non-human monoclonalantibodies (e.g., murine monoclonal antibodies) typically lack humaneffector functionality, i.e., they are unable to, inter alia, mediatecomplement dependent lysis or lyse human target cells through antibodydependent cellular toxicity or Fc-receptor mediated phagocytosis.

Chimeric antibodies comprising portions of antibodies from two or moredifferent species (e.g., mouse and human) have been developed as analternative to “conjugated” antibodies. For example, U.S. Pat. No.5,891,996 (Mateo de Acosta del Rio et al.) discusses a mouse/humanchimeric antibody, R3, directed against EGFR, and U.S. Pat. No.5,558,864 discusses generation of chimeric and humanized forms of themurine anti-EGFR MAb 425. Also, IMC-C225 (Erbitux®; ImClone) is achimeric mouse/human anti-EGFR monoclonal antibody (based on mouse M225monoclonal antibody, which resulted in HAMA responses in human clinicaltrials) that has been reported to demonstrate antitumor efficacy invarious human xenograft models. (Herbst and Shin, Cancer 94:1593-1611(2002)). The efficacy of IMC-C225 has been attributed to severalmechanisms, including inhibition of cell events regulated by EGFRsignaling pathways, and possibly by increased antibody-dependentcellular toxicity (ADCC) activity (Herbst and Shin, Cancer 94:1593-1611(2002)). IMC-C225 was also used in clinical trials, including incombination with radiotherapy and chemotherapy (Herbst and Shin, Cancer94:1593-1611 (2002)). Recently, Abgenix, Inc. (Fremont, Calif.)developed ABX-EGF for cancer therapy. ABX-EGF is a fully human anti-EGFRmonoclonal antibody. (Yang et al., Crit. Rev. Oncol./Hematol. 38:17-23(2001)).

Antibody Glycosylation

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein fuiction can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins et al.,Nature Biotechnol. 14:975-81 (1996)).

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. (Cumming et al.,Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol.14:975-81(1996)). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the blood stream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum-free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NS0- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins et al., Nature Biotechnol.14:975-81 (1996)).

All antibodies contain carbohydrate structures at conserved positions inthe heavy chain constant regions, with each isotype possessing adistinct array of N-linked carbohydrate structures, which variablyaffect protein assembly, secretion or functional activity. (Wright, A.,and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). The structure ofthe attached N-linked carbohydrate varies considerably, depending on thedegree of processing, and can include high-mannose, multiply-branched aswell as biantennary complex oligosaccharides. (Wright, A., and Morrison,S. L., Trends Biotech. 15:26-32 (1997)). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a particular glycosylation site such that even monoclonal antibodiesexist as multiple glycoforms. Likewise, it has been shown that majordifferences in antibody glycosylation occur between cell lines, and evenminor differences are seen for a given cell line grown under differentculture conditions. (Lifely, M. R. et al., Glycobiology 5(8):813-22(1995)).

One way to obtain large increases in potency, while maintaining a simpleproduction process and potentially avoiding significant, undesirableside effects, is to enhance the natural, cell-mediated effectorfunctions of monoclonal antibodies by engineering their oligosaccharidecomponent as described in Umaña, P. et al., Nature Biotechnol.17:176-180 (1999) and U.S. Pat. No. 6,602,684, the contents of which arehereby incorporated by reference in their entirety. IgG1 typeantibodies, the most commonly used antibodies in cancer immunotherapy,are glycoproteins that have a conserved N-linked glycosylation site atAsn297 in each CH2 domain. The two complex biantennary oligosaccharidesattached to Asn297 are buried between the CH2 domains, forming extensivecontacts with the polypeptide backbone, and their presence is essentialfor the antibody to mediate effector functions such as antibodydependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al.,Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol Rev.163:59-76 (1998); Wright, A. and Morrison, S. L., Trends Biotechnol.15:26-32 (1997)).

Umaña et al. showed previously that overexpression in Chinese hamsterovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III(“GnTIII”), a glycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umaña, P. et al., Nature Biotechnol.17:176-180 (1999); and International Publication No. WO 99/54342, theentire contents of which are hereby incorporated by reference). Theantibody chCE7 belongs to a large class of unconjugated mAbs which havehigh tumor affinity and specificity, but have too little potency to beclinically useful when produced in standard industrial cell lineslacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol.17:176-180 (1999)). That study was the first to show that largeincreases of ADCC activity could be obtained by engineering theantibody-producing cells to express GnTIII, which also led to anincrease in the proportion of constant region (Fc)-associated, bisectedoligosaccharides, including bisected, nonfucosylated oligosaccharides,above the levels found in naturally-occurring antibodies.

There remains a need for enhanced therapeutic approaches targeting EGFRfor the treatment of cell proliferation disorders in primates,including, but not limited to, humans, wherein such disorders arecharacterized by EGFR expression, particularly abnormal expression(e.g., overxpression) including, but not limited to, cancers of thebladder, brain, head and neck, pancreas, lung, breast, ovary, colon,prostate, and kidney.

BRIEF SUMMARY OF THE INVENTION

Recognizing the tremendous therapeutic potential of antigen bindingmolecules (ABMs) that have the binding specificity of the rat ICR62antibody (e.g., bind the same epitope) and that have beenglycoengineered to enhance Fc receptor binding affinity and effectorfunction, the present inventors developed a method for producing suchABMs. Inter alia, this method involves producing recombinant, chimericantibodies or chimeric fragments thereof. The efficacy of these ABMs isfurther enhanced by engineering the glycosylation profile of theantibody Fc region.

Accordingly, in one aspect, the invention is directed to an isolatedpolynucleotide comprising: (a) a sequence selected from a groupconsisting of: SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQ ID NO:124; (b) asequence selected from a group consisting of: SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, andSEQ ID NO:126; and (c) SEQ ID NO:108. In another aspect, the inventionis directed to an isolated polynucleotide comprising (a) a sequenceselectd from the group consisting of SEQ ID NO:112 and SEQ ID NO:114;(b) a sequence selectd from the group consisting of SEQ ID NO:116 andSEQ ID NO:118; and (c) SEQ ID NO:119. In one embodiment, any of thesepolynucleotides encodes a fusion polypeptide.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence selected from the group consistingof SEQ ID No:2; SEQ ID No:4; SEQ ID No:6; SEQ ID No:8; SEQ ID No:10; SEQID No:12; SEQ ID No:14; SEQ ID No:16; SEQ ID No:18; SEQ ID No:20; SEQ IDNo:22; SEQ ID No:24; SEQ ID No:26; SEQ ID No:28; SEQ ID No:30; SEQ IDNo32; SEQ ID No:34; SEQ ID No:36; SEQ ID No:38; SEQ ID No:40 and SEQ IDNo:120. In another aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence selected from the group consistingof SEQ ID No:44; SEQ ID No:46; SEQ ID No:50; and SEQ ID No:52. In oneembodiment, such polynucleotides encode fusion polypeptides.

The invention is further directed to an isolated polynucleotidecomprising a sequence having at least 80%, 85%, 90%, 95%, or 99%identity to a sequence selected from the group consisting of SEQ IDNo:2; SEQ ID No:4; SEQ ID No:6; SEQ ID No:8; SEQ ID No:10; SEQ ID No:12;SEQ ID No:14; SEQ ID No:16; SEQ ID No:18; SEQ ID No:20; SEQ ID No:22;SEQ ID No:24; SEQ ID No:26; SEQ ID No:28; SEQ ID No:30; SEQ ID No32; SEQID No:34; SEQ ID No:36; SEQ ID No:38; SEQ ID No:40 and SEQ ID No:120,wherein said isolated polynucleotide encodes a fusion polypeptide. In anadditional aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence having at least 80% identity to asequence selected from the group consisting of SEQ ID No:44; SEQ IDNo:46; SEQ ID No:50; and SEQ ID No:52, wherein said isolatedpolynucleotide encodes a fusion polypeptide.

The invention is also directed to an isolated polynucleotide encoding achimeric polypeptide having the sequence of SEQ ID No:1. In oneembodiment, the polynucleotide comprises a sequence encoding apolypeptide having the sequence of SEQ ID No:1; and a sequence encodinga polypeptide having the sequence of an antibody Fc region, or afragment thereof, from a species other than rat. The invention is alsodirected to an isolated polynucleotide encoding a chimeric polypeptidehaving a sequence selected from the group consisting of SEQ ID No:3; SEQID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ IDNo:15; SEQ ID No:17; SEQ ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ IDNo:25; SEQ ID No:27; SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ IDNo:35; SEQ ID No:37; SEQ ID No:39; and SEQ ID No:121. In one embodiment,the polynucleotide comprises a sequence encoding a polypeptide having asequence selected from the group consisting of SEQ ID No:3; SEQ ID No:5;SEQ ID No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQID No:17; SEQ ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ IDNo:27; SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ IDNo:37; SEQ ID No:39; and SEQ ID No:121; and a sequence encoding apolypeptide having the sequence of an antibody Fc region, or a fragmentthereof, from a species other than rat.

In yet another aspect, the invention is directed to an isolatedpolynucleotide encoding a chimeric polypeptide having the sequence ofSEQ ID No:43. In one embodiment, the polynucleotide comprises a sequenceencoding a polypeptide having the sequence of SEQ ID No:43; and asequence encoding a polypeptide having the sequence of an antibody Fcregion, or a fragment thereof, from a species other than rat. In yetanother aspect, the invention is directed to an isolated polynucleotideencoding a chimeric polypeptide having a sequence selected from thegroup consisting of SEQ ID No:45; SEQ ID No:49; and SEQ ID No:51. In oneembodiment, the polynucleotide comprises a sequence encoding apolypeptide having a sequence selected from the group consisting of SEQID No:45; SEQ ID No:49; and SEQ ID No:51, and a sequence encoding apolypeptide having the sequence of an antibody light chain constantregion (CL), or a fragment thereof, from a species other than rat.

The invention is also directed to an isolated polynucleotide comprisinga sequence encoding a polypeptide having the VH region of the ICR62antibody, or functional variants thereof, and a sequence encoding apolypeptide having the sequence of an antibody Fc region, or a fragmentthereof, from a species other than rat. In another aspect, the inventionis directed to an isolated polynucleotide comprising a sequence encodinga polypeptide having the VL region of the ICR62 antibody, or functionalvariants thereof, and a sequence encoding a polypeptide having thesequence of an antibody CL region, or a fragment thereof, from a speciesother than rat.

The invention is further directed to an expression vector comprising anyof the isolated polynucleotides described above, and to a host cell thatcomprises such an expression vector. In a further aspect, the inventionis directed to a host cell comprising any of the isolatedpolynucleotides described above.

In one aspect, the invention is directed to an isolated polypeptidecomprising: (a) a sequence selected from a group consisting of: SEQ IDNO:53 SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:123, and SEQ ID NO:125; (b) a sequence selected from agroup consisting of: SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, and SEQ ID NO:127; and (c) SEQ IDNO:107. wherein said polypeptide is a fusion polypeptide.

In another aspect, the invention is directed to an isolated polypeptidecomprising (a) a sequence selected from the group consisting of SEQ IDNO:111 and SEQ ID NO:113; (b) SEQ ID NO:115; and (c) SEQ ID NO:117,wherein said polypeptide is a fusion polypeptide.

The invention is also directed to a chimeric polypeptide comprising thesequence of SEQ ID NO:1 or a variant thereof. The invention is furtherdirected to a chimeric polypeptide comprising the sequence of SEQ IDNO:43 or a variant thereof. In one embodiment, any one of thesepolypeptides further comprises a human Fc region and/or a human CLregion. The invention is also directed to a chimeric polypeptidecomprising a sequence selected from the group consisting of SEQ ID No:3;SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQID No:15; SEQ ID No:17; SEQ ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ IDNo:25; SEQ ID No:27; SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ IDNo:35; SEQ ID No:37; SEQ ID No:39; and SEQ ID No: 121, or a variantthereof. The invention is further directed to a chimeric polypeptidecomprising s sequence selected from the group consisting of SEQ IDNo:45; SEQ ID No:49; and SEQ ID No:51, or a variant thereof. In oneembodiment, any one of these polypeptides further comprises a human Fcregion and/or a human CL region. In one embodiment, the human Fc regioncomprises IgG1.

In another aspect the invention is directed to a polypeptide comprisinga sequence derived from the ICR62 antibody and a sequence derived from aheterologous polypeptide and to an antigen-binding molecule comprisingsuch a polypeptide. In one embodiment the antigen-binding molecule is anantibody. In a preferred embodiment, the antibody is chimeric. Inanother preferred embodiment, the antibody is humanized or primatized.

In another aspect, the invention is directed to an ABM, which is capableof competing with the rat ICR62 antibody for binding to EGFR and whichis chimeric. In one embodiment, the ABM is an antibody or a fragmentthereof. In a further embodiment, the ABM is a recombinant antibodycomprising a VH region having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7;SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ IDNo:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID No:37; SEQ IDNo:39; and SEQ ID No:121. In another embodiment, the ABM is arecombinant antibody comprising a VL region having an amino acidsequence selected from the group consisting of SEQ ID NO:43, SEQ IDNo:45; SEQ ID No:49; and SEQ ID No:51. In a further embodiment the ABMis a recombinant antibody that is primatized. In yet a furtherembodiment the ABM is a recombinant antibody that is humanized. Inanother embodiment, the ABM is a recombinant antibody comprising a humanFc region. In a further embodiment, any of the ABMs discussed above maybe conjugated to a moiety such as a toxin or a radiolabel.

The invention is further related to an ABM of the present invention,said ABM having modified oligosaccharides. In one embodiment themodified oligosaccharides have reduced fucosylation as compared tonon-modified oligosaccharides. In other embodiments, the modifiedoligosaccharides are hybrid or complex. In a further embodiment, the ABMhas an increased proportion of nonfucosylated oligosaccharides orbisected, nonfucosylated oligosaccharides in the Fc region of saidmolecule. In one embodiment, the bisected, nonfucosylatedoligosaccharides are hybrid. In a further embodiment, the bisected,nonfucosylated oligosaccharides are complex. In a one embodiment, atleast 20% of the oligosaccharides in the Fc region of said polypeptideare nonfucosylated or bisected, nonfucosylated. In more preferredembodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%or more of the oligosaccharides are nonfucosylated or bisected,nonfucosylated.

The invention is further related to a polynucleotide encoding any of theABMs discussed above, and to expression vectors and cells comprisingsuch a polynucleotide.

The invention is further related to a method of producing an ABM, whichis capable of competing with the rat ICR62 antibody for binding to EGFRand wherein said ABM is chimeric; said method comprising: (a) culturinga host cell comprising a polynucleotide that encodes an ABM of thepresent invention in a medium under conditions allowing the expressionof said polynucleotide encoding said ABM; and (b) recovering said ABMfrom the resultant culture.

In another aspect, the invention is related to a pharmaceuticalcomposition comprising the ABM of the invention. It is contemplated thatthe pharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier, an adjuvant or a combination thereof.

In a further aspect, the invention is related to a method of treating adisease characterized by expression of EGFR (e.g., abnormal oroverexpression of EGFR). The method comprises administering atherapeutically effective amount of the ABM of the present invention toa subject, preferably a mammalian subject, and more preferably a humanin need thereof. In a preferred embodiment, the disease is treated byadministering an ABM that is a chimeric (e.g. humanized) antibody, or achimeric fragment of an antibody. In one embodiment, the ABM isadministered in an amount of about 1.0 mg/kg to about 15.0 mg/kg. Inanother embodiment, the ABM is administered in an amount of about 1.5mg/kg to about 12.0 mg/kg. In a further embodiment, the ABM isadministered in an amount of about 1.5 mg/kg to about 4.5 mg/kg. In afurther embodiment, the ABM is adminstered in an amount of about 4.5mg/kg to about 12.0 mg/kg. In a further embodiment, the ABM isadministered in an amount selected from the group consisting of about1.5, about 4.5, and about 12.0 mg/kg.

In yet another aspect, the invention is related to a host cellengineered to express at least one nucleic acid encoding a polypeptidehaving GnTIII activity in an amount sufficient to modify theoligosaccharides in the Fc region of the ABM produced by the host cell,wherein the ABM is capable of competing with the rat ICR62 antibody forbinding to EGFR and wherein the ABM is chimeric. In one embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide. In anotherembodiment, the ABM produced by the host cell is an antibody or anantibody fragment. In one embodiment, the antibody or antibody fragmentis humanized. In a further embodiment, the ABM comprises a regionequivalent to the Fc region of a human IgG.

The invention is also directed to an isolated polynucleotide comprisingat least one (e.g., one, two, three, four, five, or six) complementaritydetermining region of the rat ICR62 antibody, or a variant or truncatedform thereof containing at least the specificity-determining residuesfor said complementarity determining region, wherein said isolatedpolynucleotide encodes a fusion polypeptide. Preferably, such isolatedpolynucleotides encode a fusion polypeptide that is an antigen bindingmolecule. In one embodiment, the polynucleotide comprises threecomplementarity determining regions of the rat ICR62 antibody, orvariants or truncated forms thereof containing at least thespecificity-determining residues for each of said three complementaritydetermining regions. In another embodiment, the polynucleotide encodesthe entire variable region of the light or heavy chain of a chimeric(e.g., humanized) antibody. The invention is further directed to thepolypeptides encoded by such polynucleotides.

In another embodiment, the invention is directed to an antigen bindingmolecule comprising at least one (e.g., one, two, three, four, five, orsix) complementarity determining region of the rat ICR62 antibody, or avariant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion, and comprising a sequence derived from a heterologouspolypeptide. In one embodiment, the antigen binding molecule comprisesthree complementarity determining regions of the rat ICR62 antibody, orvariants or truncated forms thereof containing at least thespecificity-determining residues for each of said three complementaritydetermining regions. In another aspect, the antigen binding moleculecomprises the variable region of an antibody light or heavy chain. Inone particularly useful embodiment, the antigen binding molecule is achimeric, e.g., humanized, antibody. The invention is also directed tomethods of making such antigen binding molecules, and the use of same inthe treatment of disease, including malignancies such as cancers of thebladder, brain, head and neck, pancreas, lung, breast, ovary, colon,prostate, skin, and kidney.

The host cell of the present invention may be selected from the groupthat includes, but is not limited to, an HEK293-EBNA cell, a CHO cell, aBHK cell, a NS0 cell, a SP2/0 cell, a YO myeloma cell, a P3X63 mousemyeloma cell, a PER cell, a PER.C6 cell or a hybridoma cell. In oneembodiment, the host cell of the invention further comprises atransfected polynucleotide comprising a polynucleotide encoding the VLregion of the rat ICR62 antibody or variants thereof and a sequenceencoding a region equivalent to the Fc region of a human immunoglobulin.In another embodiment, the host cell of the invention further comprisesa transfected polynucleotide comprising a polynucleotide encoding the VHregion of the rat ICR62 antibody or variants thereof and a sequenceencoding a region equivalent to the Fc region of a human immunoglobulin.

In a further aspect, the invention is directed to a host cell thatproduces an ABM that exhibits increased Fc receptor binding affinityand/or increased effector function as a result of the modification ofits oligosaccharides. In one embodiment, the increased binding affinityis to an Fc receptor, particularly, the FcγRIIIA receptor. The effectorfunction contemplated herein may be selected from the group thatincludes, but is not limited to, increased Fc-mediated cellularcytotoxicity; increased binding to NK cells; increased binding tomacrophages; increased binding to polymorphonuclear cells; increasedbinding to monocytes; increased direct signaling inducing apoptosis;increased dendritic cell maturation; and increased T cell priming.

In a further embodiment, the host cell of the present inventioncomprises at least one nucleic acid encoding a polypeptide having GnTIIIactivity that is operably linked to a constitutive promoter element.

In another aspect, the invention is directed to a method for producingan ABM in a host cell, comprising: (a) culturing a host cell engineeredto express at least one polynucleotide encoding a fusion polypeptidehaving GnTIII activity under conditions which permit the production ofsaid ABM and which permit the modification of the oligosaccharidespresent on the Fc region of said ABM; and (b) isolating said ABM;wherein said ABM is capable of competing with the rat ICR62 antibody forbinding to EGFR and wherein said ABM is chimeric (e.g., humanized). Inone embodiment, the polypeptide having GnTIII activity is a fusionpolypeptide, preferably comprising the catalytic domain of GnTIII andthe Golgi localization domain of a heterologous Golgi residentpolypeptide selected from the group consisting of the localizationdomain of mannosidase II, the localization domain ofβ(1,2)-N-acetylglucosaminyltransferase I (“GnTI”), the localizationdomain of mannosidase I, the localization domain ofβ(1,2)-N-acetylglucosaminyltransferase II (“GnTII”), and thelocalization domain of α1-6 core fucosyltransferase. Preferably, theGolgi localization domain is from mannosidase II or GnTI.

In a further aspect, the invention is directed to a method for modifyingthe glycosylation profile of an anti-EGFR ABM produced by a host cellcomprising introducing into the host cell at least one nucleic acid orexpression vector of the invention. In one embodiment, the ABM is anantibody or a fragment thereof; preferably comprising the Fc region ofan IgG. Alternatively, the polypeptide is a fusion protein that includesa region equivalent to the Fc region of a human IgG.

In one aspect, the invention is related to a recombinant, chimericantibody, or a fragment thereof, capable of competing with the rat ICR62antibody for binding to EGFR and having reduced fucosylation.

In another aspect, the present invention is directed to a method ofmodifying the glycosylation of the recombinant antibody or a fragmentthereof of the invention by using a fusion polypeptide having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide. In one embodiment, the fusion polypeptidesof the invention comprise the catalytic domain of GnTIII. In anotherembodiment, the Golgi localization domain is selected from the groupconsisting of: the localization domain of mannosidase II, thelocalization domain of GnTI, the localization domain of mannosidase I,the localization domain of GnTII and the localization domain of α1-6core fucosyltransferase. Preferably, the Golgi localization domain isfrom mannosidase II or GnTI.

In one embodiment, the method of the invention is directed towardsproducing a recombinant, chimeric antibody or a fragment thereof, withmodified oligosaccharides wherein said modified oligosaccharides havereduced fucosylation as compared to non-modified oligosaccharides.According to the present invention, these modified oligosaccharides maybe hybrid or complex. In another embodiment, the method of the inventionis directed towards producing a recombinant, chimeric (e.g., humanized)antibody or a fragment thereof having an increased proportion ofbisected, nonfucosylated oligosaccharides in the Fc region of saidpolypeptide. In one embodiment, the bisected, nonfucosylatedoligosaccharides are hybrid. In another embodiment, the bisected,nonfucosylated oligosaccharides are complex. In a further embodiment,the method of the invention is directed towards producing a recombinant,chimeric antibody or a fragment thereof having at least 20% of theoligosaccharides in the Fc region of said polypeptide that are bisected,nonfucosylated. In a preferred embodiment, at least 30% of theoligosaccharides in the Fc region of said polypeptide are bisected,nonfucosylated. In another preferred embodiment, wherein at least 35% ofthe oligosaccharides in the Fc region of said polypeptide are bisected,nonfucosylated.

In a further aspect, the invention is directed to a recombinant,chimeric antibody or a fragment thereof, that exhibits increased Fcreceptor binding affinity and/or increased effector function as a resultof the modification of its oligosaccharides. In one embodiment, theincreased binding affinity is to an Fc activating receptor. In a furtherembodiment, the Fc receptor is Fcγ activating receptor, particularly,the FcγRIIIA receptor. The effector function contemplated herein may beselected from the group that includes, but is not limited to, increasedFc-mediated cellular cytotoxicity; increased binding to NK cells;increased binding to macrophages; increased binding to polymorphonuclearcells; increased binding to monocytes; increased direct signalinginducing apoptosis; increased dendritic cell maturation; and increased Tcell priming.

In another aspect, the invention is directed to a recombinant, chimeric(e.g., humanized) antibody fragment, having the binding specificity ofthe rat ICR62 antibody and containing the Fc region, that is engineeredto have increased effector function produced by any of the methods ofthe present invention.

In another aspect, the present invention is directed to a fusion proteinthat includes a polypeptide having a sequence selected from the groupconsisting of of SEQ ID NO: 1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7;SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ IDNo:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID No:37; SEQ IDNo:39; and SEQ ID No:121, and a region equivalent to the Fc region of animmunoglobulin and engineered to have increased effector functionproduced by any of the methods of the present invention.

In another aspect, the present invention is directed to a fusion proteinthat includes a polypeptide having a sequence selected from the groupconsisting of SEQ ID NO:43, SEQ ID No:45; SEQ ID No:49; and SEQ ID No:51and a region equivalent to the Fc region of an immunoglobulin andengineered to have increased effector function produced by any of themethods of the present invention.

In one aspect, the present invention is directed to a pharmaceuticalcomposition comprising a recombinant, chimeric (e.g., humanized)antibody, produced by any of the methods of the present invention, and apharmaceutically acceptable carrier. In another aspect, the presentinvention is directed to a pharmaceutical composition comprising arecombinant, chimeric (e.g., humanized) antibody fragment produced byany of the methods of the present invention, and a pharmaceuticallyacceptable carrier. In another aspect, the present invention is directedto a pharmaceutical composition comprising a fusion protein produced byany of the methods of the present invention, and a pharmaceuticallyacceptable carrier.

In a further aspect, the invention is directed to a method fortargetting in vivo or in vitro cells expressing EGFR. In one embodiment,the present invention is directed to a method for targetting cellsexpressing EGFR in a subject comprising administering to the subject acomposition comprising an ABM of the invention.

In yet another aspect, the present invention is directed to a method fordetecting in vivo or in vitro the presence of EGFR in a sample, e.g.,for diagnosing a disorder related to EGFR expression. In one embodiment,the detection is performed by contacting a sample to be tested,optionally with a control sample, with an ABM of the present invention,under conditions that allow for formation of a complex between the ABMand EGFR. The complex formation is then detected (e.g., by ELISA orother methods known in the art). When using a control sample with thetest sample, any statistically significant difference in the formationof ABM-EGFR complexes when comparing the test and control samples isindicative of the presence of EGFR in the test sample.

The invention is further directed to a method of treating a disorderrelated to EGFR expression, in particular, a cell proliferation disorderwherein EGFR is expressed, and more particularly, wherein EGFR isabnormally expressed (e.g. overexpressed), including cancers of thebladder, brain, head and neck, pancreas, lung, breast, ovary, colon,prostate, skin, and kidney comprising administering a therapeuticallyeffective amount of the recombinant, chimeric (e.g., humanized) antibodyor fragment thereof, produced by any of the methods of the presentinvention, to a human subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the functional activity of individual heavy and lightchimeric rat-human ICR62 polypeptide chains when combined with thehumanized ICR62 constructs I-HHC (heavy chain) and I-KB (light chain).rVL represents the chimeric light chain, and rVH represents the chimericheavy chain. The “r” designation indicates that the variable domains arefrom the original rat antibody.

FIG. 2 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HHC, I-HHA and I-HLA andhumanized light chain variable region constructs I-KA and I-KB paired invarious configurations.

FIG. 3 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HLB, I-HLC and I-HLA andhumanized light chain variable region constructs I-KA and I-KC paired invarious configurations.

FIG. 4 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HLA2, I-HLA3 and I-HLA4 andhumanized light chain variable region construct I-KC as compared tochimeric rat-human ICR62 antibody.

FIG. 5 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HLA1, I-HLA3, I-HLA5 and I-HLA6and humanized light chain variable region construct I-KC as compared tochimeric rat-human ICR62 antibody.

FIG. 6 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HLA7, I-HLA6, and I-HHB andhumanized light chain variable region construct I-KC as compared tochimeric rat-human ICR62 antibody.

FIG. 7 shows binding activity of humanized ICR62 antibodies comprisingheavy chain variable region constructs I-HHF, I-HLA9, and I-HLA8 andhumanized light chain variable region construct I-KC as compared tochimeric rat-human ICR62 antibody.

FIG. 8 shows binding activity of humanized antibodies comprising heavychain varible region constructs I-HHB, I-HHD, I-HHG, I-HHF, I-HLA7, andI-HLA9 and humanized light chain variable region construct I-KC.

FIG. 9 shows a comparison of antibody mediated cellular cytotoxicity(ADCC) for various glycoforms of the chimeric ICR62 antibody, as well asfor the humanized variant I-HLA4. “G1” refers to glcyoengineering of theantibody by co-expression with GnTIII. “G2” refers to glycoengineeringof the antibody by co-expression with GnTIII and ManII. “WT” refers toantibodies that were not glycoengineered. The humanzied heavy chainconstructs were paired with the I-KC light chain construct.

FIG. 10 shows a comparison of ADCC for the non-glycoengineered form (WT)and the G2 glycoform (i.e., glycoengineered by co-expression with GnTIIIand ManII) of the humanized ICR62 antibody constructs I-HHB and I-HLA7.The same antibodies were applied to two different target cell lines: inPanel A, the target cell line LN229 was used; in Panel B, the cell lineA431 was used. The humanzied heavy chain constructs were paired with theI-KC light chain construct.

FIG. 11A and 11B show a comparison of ADCC for non-glycoengineered forms(WT) and G2 glcyoforms of chimeric ICR62 and the humanized ICR62antibody constructs I-HHB and I-HLA7. The target cell line A431 wasused. The humanzied heavy chain constructs were paired with the I-KClight chain construct.

FIG. 12 shows a comparison of 72h ADCC for G2 glcyoforms of chimericICR62 and the humanized ICR62 antibody constructs I-HHB and I-HLA7. Thehumanzied heavy chain constructs were paired with the I-KC light chainconstruct.

FIG. 13 shows an amino acid sequence alignment of humanized ICR62 heavychain variable region constructs compared to the rat ICR62 sequences.Dots represent identity of amino acid residues at a given positionwithin a given construct.

FIG. 14 shows an FcgammaRIIIa-Fc binding assay using CHO cellsdisplaying recombinant human FcgammaRIIIa. A glycoengineered I-HHB/KChumanized anti-EGFR IgG1 antibody was compared to a non-glycoengineered(Wt) antibody.

FIG. 15 shows a MALD/TOF-MS oligosaccharide profile for glycoengineeredhumanized anti-EGFR IgG1 antibody, I-HHB/KC. Glycoengineering achievedby overexpression in the antibody-producing cells of genes encodingenzymes with GnTIII and Golgi Mannosidase II activities, yielding over70% of non-fucosylated Fc-Asn297-linked oligosaccharides.

FIG. 16 shows an anti-EGFR precision profile (n=6 replicates across thecalibration range) for the determination of anti-EGFR in 1% monkey serummatrix (monkey serum pool CMS25/31/33, supplied by HLS).

FIG. 17 shows a representative anti-EGFR calibration curve for thedetermination of anti-EGFR in 1% monkey serum matrix.

FIG. 18 shows serum concentrations of anti-EGFR on Day 1 of weeklyintravenous administration of anti-EGFR to male cynomolgous monkeys.

FIG. 19 shows serum concentrations of anti-EGFR on Day 1 of weeklyintravenous administration of anti-EGFR to female cynomolgous monkeys.

FIG. 20 shows the relationship between areas under the serum anti-EGFRconcentration-time curves (AUC₁₆₈) and dose level on Day 1 of weeklyintravenous administration of anti-EGFR to cynomolgous monkeys.

FIG. 21 shows serum concentrations of anti-EGFR during weeklyintravenous administration of anti-EGFR to male cynomolgous monkeys.

FIG. 22 shows serum concentrations of anti-EGFR during weeklyintravenous administration of anti-EGFR to female cynomolgous monkeys.

FIG. 23 shows the MALDI/TOF-MS profile of oligosaccharides fromFc-engineered (glycoengineered) anti-EGFR antibody used for the in vivomonkey studies described in the Examples herein below.

FIG. 24 shows binding to EGFR expressed on the surface of human A431epidermoid carcinoma cells. The antibody used for the binding study wasthe Fc-engineered anti-EGFR antibody (I-HHB construct) used for the invivo monkey studies described in the Examples herein below.

FIG. 25 shows binding to EGFR expressed on surface of monkey COS-7kidney cells. The antibody used was anti-EGFR antibody (I-HHB heavychain; I-KC light chain). For reference, binding to low humanEGFR-expressing cells, MCF-7 breast cancer cells, is shown.

FIG. 26 shows Fc-FcgammaRIIIa binding using a whole cell (CHO cellsengineered to express human FcgRIIIa on their surface). The antibodyused was the Fc-engineered (glycoengineered) anti-EGFR antibody used forthe in vivo monkey studies described in the Examples herein below.Binding for a non-Fc-engineered (unmodified) control IgG1 antibody isshown for comparison.

FIG. 27 shows ADCC mediated by Fc-engineered (glycoengineered) anti-EGFRantibody. Target cells are A549 human lung carcinoma cells. ADCCactivity for the non-Fc engineered (unmodified) form of the antibody isshown for comparison.

FIG. 28 shows ADCC mediated by Fc-engineered (glycoengineered) anti-EGFRantibody. Target cells are CYNOM-K1 cynomolgus monkey keratinocyte cellline. ADCC activity for the non-Fc engineered (unmodified) form of theantibody is shown for comparison.

FIG. 29 shows EGFR target binding of various light chain constructvariants based on the I-KC construct paired with the heavy chain I-HHDconstruct.

DETAILED DESCRIPTION OF THE INVENTION

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, the term antibody is intended to include whole antibodymolecules, including monoclonal, polyclonal and multispecific (e.g.,bispecific) antibodies, as well as antibody fragments having the Fcregion and retaining binding specificity, and fusion proteins thatinclude a region equivalent to the Fc region of an immunoglobulin andthat retain binding specificity. Also encompassed are antibody fragmentsthat retain binding specificity including, but not limited to, VHfragments, VL fragments, Fab fragments, F(ab′)₂ fragments, scFvfragments, Fv fragments, minibodies, diabodies, triabodies, andtetrabodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134(2003)). Also encompassed are humanized, primatized and chimericantibodies.

As used herein, the term Fc region is intended to refer to a C-terninalregion of an IgG heavy chain. Although the boundaries of the Fc regionof an IgG heavy chain might vary slightly, the human IgG heavy chain Fcregion is usually defined to stretch from the amino acid residue atposition Cys226 to the carboxyl-terminus.

As used herein, the term region equivalent to the Fc region of animmunoglobulin is intended to include naturally occurring allelicvariants of the Fc region of an immunoglobulin as well as variantshaving alterations which produce substitutions, additions, or deletionsbut which do not decrease substantially the ability of theimmunoglobulin to mediate effector functions (such as antibody dependentcellular cytotoxicity). For example, one or more amino acids can bedeleted from the N-terminus or C-terminus of the Fc region of anirnmunoglobulin without substantial loss of biological function. Suchvariants can be selected according to general rules known in the art soas to have minimal effect on activity. (See, e.g., Bowie, J. U. et al.,Science 247:1306-1310 (1990).

As used herein, the term EGFR refers to the human epidermal growthfactor receptor (also known as HER-1 or Erb-B1) (Ulrich, A. et al.,Nature 309:418-425 (1984); SwissProt Accession #P00533; secondaryaccession numbers: O00688, O00732, P06268, Q14225, Q92795, Q9BZS2,Q9GZX1, Q9H2C9, Q9H3C9, Q9UMD7, Q9UMD8, Q9UMG5), as well asnaturally-occurring isoforms and variants thereof. Such isoforms andvariants include but are not limited to the EGFRvIII variant,alternative splicing products (e.g., as identified by SwissProtAccession numbers P00533-1, P00533-2, P00533-3, P00533-4), variantsGLN-98, ARG-266, Lys-521, ILE-674, GLY-962, and PRO-988 (Livingston, R.J. et al., NIEHS-SNPs, environmental genome project, NIEHS ES15478,Department of Genome Sciences, Seattle, Wash. (2004)), and othersidentified by the following accession numbers: NM_(—)005228.3,NM_(—)201282.1, NM_(—)201283.1, NM_(—)201284.1 (REFSEQ mRNAs);AF125253.1, AF277897.1, AF288738.1, AI217671.1, AK127817.1, AL598260.1,AU137334.1, AW163038.1, AW295229.1, BC057802.1, CB160831.1, K03193.1,U48722.1, U95089.1, X00588.1, X00663.1; H54484S1, H54484S3, H54484S2(MIPS assembly); DT.453606, DT.86855651, DT.95165593, DT.97822681,DT.95165600, DT.100752430, DT.91654361, DT.92034460, DT.92446349,DT.97784849, DT.101978019, DT.418647, DT.86842167, DT.91803457,DT.92446350, DT.95153003, DT.95254161, DT.97816654, DT.87014330,DT.87079224 (DOTS Assembly).

As used herein, the term EGFR ligand refers to a polypeptide which bindsto and/or activates EGFR. The term includes membrane-bound precursorforms of the EGFR ligand, as well as proteolytically processed solubleforms of the EGFR ligand.

As used herein, the term ligand activation of EGFR refers to signaltransduction (e.g., that caused by an intracellular kinase domain ofEGFR receptor phosphorylating tyrosine residues in the EGFR or asubstrate polypeptide) mediated by EGFR ligand binding.

As used herein, the term disease or disorder characterized by abnormalactivation or production of EGFR or an EGFR ligand or disorder relatedto EGFR expression, refers to a condition, which may or may not involvemalignancy or cancer, where abnormal activation and/or production ofEGFR and/or an EGFR ligand is occurring in cells or tissues of a subjecthaving, or predisposed to, the disease or disorder.

As used herein, the terms overexpress, overexpressed, andoverexpressing, as used in connection with cells expressing EGFR, referto cells which have measurably higher levels of EGFR on the surfacethereof compared to a normal cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. EGFR expression (and, hence,overexpression) may be determined in a diagnostic or prognostic assay byevaluating levels of EGFR present on the suface of a cell or in a celllysate by techniques that are known in the art: e.g., via animmunohistochemistry assay, immunofluorescence assay, immunoenzymeassay, ELISA, flow cytometry, radioimmunoassay, Western blot, ligandbinding, kinase activity, etc. (See generally, CELL BIOLOGY: ALABORATORY HANDBOOK, Celis, J., ed., Academic Press (2d ed., 1998);CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan, J. E. et al., eds., JohnWiley & Sons (1995-2003); see also, Sumitomo et al., Clin. Cancer Res.10: 794-801 (2004) (describing Western blot, flow cytometry, andimmunohistochemstry) the entire contents of which are hereinincorporated by reference)). Alternatively, or additionally, one maymeasure levels of EGFR-encoding nucleic acid molecules in the cell,e.g., via fluorescent in situ hybridization, Southern blotting, or PCRtechniques. The levels of EGFR in normal cells are compared to thelevels of cells affected by a cell proliferation disorder (e.g., cancer)to determine if EGFR is overexpressed.

As used herein, the term antigen binding molecule refers in its broadestsense to a molecule that specifically binds an antigenic determinant.More specifically, an antigen binding molecule that binds EGFR is amolecule which specifically binds to a transmembrane receptor of 170kDa, typically designated as the epidermal growth factor receptor(EGFR), but also known as HER-1 or ErbB1. By “specifically binds” ismeant that the binding is selective for the antigen and can bediscriminated from unwanted or nonspecific interactions.

As used herein, the terms fusion and chimeric, when used in reference topolypeptides such as ABMs refer to polypeptides comprising amino acidsequences derived from two or more heterologous polypeptides, such asportions of antibodies from different species. For chimeric ABMs, forexample, the non-antigen binding components may be derived from a widevariety of species, including primates such as chimpanzees and humans.The constant region of the chimeric ABM is most preferably substantiallyidentical to the constant region of a natural human antibody; thevariable region of the chimeric antibody is most preferablysubstantially identical to that of a recombinant anti-EGFR antibodyhaving the amino acid sequence of the murine variable region. Humanizedantibodies are a particularly preferred form of fusion or chimericantibody.

As used herein, a polypeptide having GnTIII activity refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1-4 linkage to the β-linkedmannoside of the trimannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependence in agiven activity as compared to the GnTIII (i.e., the candidatepolypeptide will exhibit greater activity or not more than about 25-foldless and, preferably, not more than about tenfold less activity, andmost preferably, not more than about three-fold less activity relativeto the GnTIII.)

As used herein, the term variant (or analog) refers to a polypeptidediffering from a specifically recited polypeptide of the invention byamino acid insertions, deletions, and substitutions, created using, eg., recombinant DNA techniques. Variants of the ABMs of the presentinvention include chimeric, primatized or humanized antigen bindingmolecules wherein one or several of the amino acid residues are modifiedby substitution, addition and/or deletion in such manner that does notsubstantially affect antigen (e.g., EGFR) binding affinity. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, may be found by comparing thesequence of the particular polypeptide with that of homologous peptidesand minimizing the number of amino acid sequence changes made in regionsof high homology (conserved regions) or by replacing amino acids withconsensus sequence.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are preferably in the rangeof about 1 to 20 amino acids, more preferably 1 to 10 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

As used herein, the term humanized is used to refer to anantigen-binding molecule derived from a non-human antigen-bindingmolecule, for example, a murine antibody, that retains or substantiallyretains the antigen-binding properties of the parent molecule but whichis less immunogenic in humans. This may be achieved by various methodsincluding (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies, (b) grafting only thenon-human CDRs onto human framework and constant regions with or withoutretention of critical framework residues (e.g., those that are importantfor retaining good antigen binding affinity or antibody functions), or(c) transplanting the entire non-human variable domains, but “cloaking”them with a human-like section by replacement of surface residues. Suchmethods are disclosed in Jones et al., Morrison et al., Proc. Natl.Acad. Sci., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol.,44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988);Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,31(3):169-217 (1994), all of which are incorporated by reference intheir entirety herein. There are generally 3 complementarity determiningregions, or CDRs, (CDR1, CDR2 and CDR3) in each of the heavy and lightchain variable domains of an antibody, which are flanked by fourframework subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavyand light chain variable domains of an antibody:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of humanized antibodies canbe found, inter alia, in U.S. Pat. No. 6,632,927, and in published U.S.Application No. 2003/0175269, both of which are incorporated herein byreference in their entirety.

Similarly, as used herein, the term primatized is used to refer to anantigen-binding molecule derived from a non-primate antigen-bindingmolecule, for example, a murine antibody, that retains or substantiallyretains the antigen-binding properties of the parent molecule but whichis less immunogenic in primates.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table I as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody. TABLE 1 CDR Definitions¹ Kabat Chothia AbM²V_(H) CDR1 31-35 26-32 26-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3 95-102  95-102  95-102 V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-5650-52 50-56 V_(L) CDR3 89-97 91-96 89-97¹Numbering of all CDR definitions in Table 1 is according to thenumbering conventions set forth by Kabat et al. (see below).²“AbM” refers to the CDRs as defined by Oxford Molecular's “AbM”antibody modeling software.

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambigously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an ABM are according to the Kabatnumbering system. The sequences of the sequence listing (i.e., SEQ IDNO:1 to SEQ ID NO:127) are not numbered according to the Kabat numberingsystem. However, as stated above, it is well within the ordinary skillof one in the art to determine the Kabat numbering scheme of anyvariable region sequence in the Sequence Listing based on the numberingof the sequences as presented therein.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide sequence of thepresent invention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencepolypeptide can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

As used herein, a nucleic acid that “hybridizes under stringentconditions” to a nucleic acid sequence of the invention, refers to apolynucleotide that hybridizes in an overnight incubation at 42° C. in asolution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

As used herein, the term Golgi localization domain refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide in location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the term effector function refers to those biologicalactivities attributable to the Fc region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector finctions include, but are not limited to, Fc receptorbinding affinity, antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune-complex-mediated antigen uptake by antigen-presenting cells,down-regulation of cell surface receptors, etc.

As used herein, the terms engineer, engineered, engineering andglycosylation engineering are considered to include any manipulation ofthe glycosylation pattern of a naturally occurring or recombinantpolypeptide or fragment thereof. Glycosylation engineering includesmetabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation. In one embodiment, theglycosylation engineering is an alteration in glycosyltransferaseactivity. In a particular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.

As used herein, the term host cell covers any kind of cellular systemwhich can be engineered to generate the polypeptides and antigen-bindingmolecules of the present invention. In one embodiment, the host cell isengineered to allow the production of an antigen binding molecule withmodified glycoforns. In a preferred embodiment, the antigen bindingmolecule is an antibody, antibody fragment, or fusion protein. Incertain embodiments, the host cells have been further manipulated toexpress increased levels of one or more polypeptides having GnTIIIactivity. Host cells include cultured cells, e.g., mammalian culturedcells, such as CHO cells, HEK293-EBNA cells, BHK cells, NS0 cells, SP2/0cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6cells or hybridoma cells, yeast cells; insect cells, and plant cells, toname only a few, but also cells comprised within a transgenic animal,transgenic plant or cultured plant or animal tissue.

As used herein, the term Fc-mediated cellular cytotoxicity includesantibody-dependent cellular cytotoxicity and cellular cytotoxicitymediated by a soluble Fc-fusion protein containing a human Fc-region. Itis an immune mechanism leading to the lysis of “antibody-targeted cells”by “human immune effector cells”, wherein:

The human immune effector cells are a population of leukocytes thatdisplay Fc receptors on their surface through which they bind to theFc-region of antibodies or of Fc-fusion proteins and perform effectorfunctions. Such a population may include, but is not limited to,peripheral blood mononuclear cells (PBMC) and/or natural killer (NK)cells.

The antibody-targeted cells are cells bound by the antibodies orFc-fusion proteins. The antibodies or Fc fusion-proteins bind to targetcells via the protein part N-terminal to the Fc region.

As used herein, the term increased Fc-mediated cellular cytotoxicity isdefined as either an increase in the number of “antibody-targeted cells”that are lysed in a given time, at a given concentration of antibody, orof Fc-fusion protein, in the medium surrounding the target cells, by themechanism of Fc-mediated cellular cytotoxicity defined above, and/or areduction in the concentration of antibody, or of Fc-fusion protein, inthe medium surrounding the target cells, required to achieve the lysisof a given number of “antibody-targeted cells”, in a given time, by themechanism of Fc-mediated cellular cytotoxicity. The increase inFc-mediated cellular cytotoxicity is relative to the cellularcytotoxicity mediated by the same antibody, or Fc-fusion protein,produced by the same type of host cells, using the same standardproduction, purification, formulation and storage methods, which areknown to those skilled in the art, but that has not been produced byhost cells engineered to express the glycosyltransferase GnTIII by themethods described herein.

By antibody having increased antibody dependent cellular cytotoxicity(ADCC) is meant an antibody, as that term is defined herein, havingincreased ADCC as determined by any suitable method known to those ofordinary skill in the art. One accepted in vitro ADCC assay is asfollows:

1) the assay uses target cells that are known to express the targetantigen recognized by the antigen-binding region of the antibody;

2) the assay uses human peripheral blood mononuclear cells (PBMCs),isolated from blood of a randomly chosen healthy donor, as effectorcells;

3) the assay is carried out according to following protocol:

-   -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labeled with 100 micro-Curies of 51Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 105 cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04        ng/ml in cell culture medium and 50 microliters of the resulting        antibody solutions are added to the target cells in the 96-well        microtiter plate, testing in triplicate various antibody        concentrations covering the whole concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labeled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the antibody        solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labeled target cells, receive        50 microliters of RPMI cell culture medium instead of the        antibody solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter;    -   x) the percentage of specific lysis is calculated for each        antibody concentration according to the formula        (ER-MR)/(MR-SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that antibody concentration,        MR is the average radioactivity quantified (see point ix above)        for the MR controls (see point v above), and SR is the average        radioactivity quantified (see point ix above) for the SR        controls (see point vi above);

4) “increased ADCC” is defined as either an increase in the maximumpercentage of specific lysis observed within the antibody concentrationrange tested above, and/or a reduction in the concentration of antibodyrequired to achieve one half of the maximum percentage of specific lysisobserved within the antibody concentration range tested above. Theincrease in ADCC is relative to the ADCC, measured with the above assay,mediated by the same antibody, produced by the same type of host cells,using the same standard production, purification, formulation andstorage methods, which are known to those skilled in the art, but thathas not been produced by host cells engineered to overexpress GnTIII.

In one aspect, the present invention is related to antigen bindingmolecules having the binding specificity of the rat ICR62 (i.e., bindsto substantially the same epitope), and to the discovery that theireffector functions can be enhanced by altered glycosylation. In oneembodiment, the antigen binding molecule is a chimeric antibody. In apreferred embodiment, the invention is directed to a chimeric antibody,or a fragment thereof, comprising one or more (e.g., one, two, three,four, five, or six) of the the CDRs of any of SEQ ID NOs:53-108 and/orSEQ ID NO:s 122-127. Specifically, in a preferred embodiment, theinvention is directed to an isolated polynucleotide comprising: (a) asequence selected from a group consisting of: SEQ ID NO:54 SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122,and SEQ ID NO:124; (b) a sequence selected from a group consisting of:SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,SEQ ID NO:106, and SEQ ID NO:126; and (c) SEQ ID NO:108. In anotherpreferred embodiment, the invention is directed to an isolatedpolynucleotide comprising (a) a sequence selectd from the groupconsisting of SEQ ID NO:112 and SEQ ID NO:114; (b) a sequence selectdfrom the group consisting of SEQ ID NO:116 and SEQ ID NO:118; and (c)SEQ ID NO:119. In one embodiment, any of these polynucleotides encodes afusion polypeptide.

In another embodiment, the antigen binding molecule comprises the V_(H)domain of the rat ICR62 antibody encoded by SEQ ID NO:1 or SEQ ID NO:2,or a variant thereof; and a non-murine polypeptide. In another preferredembodiment, the invention is directed to an antigen binding moleculecomprising the V_(L) domain of the rat antibody encoded by SEQ ID NO:43or SEQ ID NO:44, or a variant thereof; and a non-murine polypeptide.

In another aspect, the invention is directed to antigen bindingmolecules comprising one or more (e.g., one, two, three, four, five, orsix) truncated CDRs of ICR62. Such truncated CDRs will contain, at aminimum, the specificity-determining amino acid residues for the givenCDR. By “specificity-determining residue” is meant those residues thatare directly involved in the interaction with the antigen. In general,only about one-fifth to one-third of the residues in a given CDRparticipate in binding to antigen. The specificity-determining residuesin a particular CDR can be identified by, for example, computation ofinteratomic contacts from three-dimensional modeling and determinationof the sequence variability at a given residue position in accordancewith the methods described in Padlan et al., FASEB J. 9(1):133-139(1995), the contents of which is hereby incorporated by reference intheir entirety.

Accordingly, the invention is also directed to an isolatedpolynucleotide comprising at least one (e.g., one, two, three, four,five, or six) complementarity determining region of the rat ICR62antibody, or a variant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion, wherein said isolated polynucleotide encodes a fusionpolypeptide. Preferably, such isolated polynucleotides encode a fusionpolypeptide that is an antigen binding molecule. In one embodiment, thepolynucleotide comprises three complementarity determining regions ofthe rat ICR62 antibody, or variants or truncated forms thereofcontaining at least the specificity-determining residues for each ofsaid three complementarity determining regions. In one embodiment, thepolynucleotide comprises at least one of the CDRs set forth in Tables2-5, below. In another embodiment, the polynucleotide encodes the entirevariable region of the light or heavy chain of a chimeric (e.g.,humanized) antibody. The invention is further directed to thepolypeptides encoded by such polynucleotides.

In another embodiment, the invention is directed to an antigen bindingmolecule comprising at least one (e.g., one, two, three, four, five, orsix) complementarity determining region of the rat ICR62 antibody, or avariant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion, and comprising a sequence derived from a heterologouspolypeptide. In one embodiment, the antigen binding molecule comprisesthree complementarity determining regions of the rat ICR62 antibody, orvariants or truncated forms thereof containing at least thespecificity-determining residues for each of said three complementaritydetermining regions. In one embodiment, the antigen binding moleculecomprises at least one of the CDRs set forth in Tables 2-5, below. Inanother aspect, the antigen binding molecule comprises the variableregion of an antibody light or heavy chain. In one particularly usefullembodiment, the antigen binding molecule is a chimeric, e.g., humanized,antibody. The invention is also directed to methods of making suchantigen binding molecules, and the use of same in the treatment ofdisease, particularly cell proliferation disorders wherein EGFR isexpressed, particularly wherein EGFR is abnormally expressed (e.g.,overexpressed) compared to normal tissue of the same cell type. Suchdisorders include, but are not limited to cancers of the bladder, brain,head and neck, pancreas, lung, breast, ovary, colon, prostate, skin, andkidney. EGFR expression levels may be determined by methods known in theart and those described herein (e.g., via immunohistochemistry assay,immunofluorescence assay, immunoenzyme assay, ELISA, flow cytometry,radioimmunoassay, Western blot, ligand binding, kinase activity, etc.).

The invention is also directed to a method for targeting in vivo or invitro cells expressing EGFR. Cells that express EGFR may be targeted fortherapuetic purposes (e.g., to treat a disorder that is treatable bydisruption of EGFR-mediated signaling, for example by blocking ligandbinding, or by targeting EGFR-expressing cells for destruction by theimmune system). In one embodiment, the present invention is directed toa method for targeting cells expressing EGFR in a subject comprisingadministering to the subject a composition comprising an ABM of theinvention. Cells that express EGFR may also be targeted for diagnositicpurposes (e.g., to determine if they are expressing EGFR, eithernormally or abnormally). Thus, the invention is also directed to methodsfor detecting the presence of EGFR or a cell expressing EGFR, either invivo or in vitro. One method of detecting EGFR expression according tothe present invention comprises contacting a sample to be tested,optionally with a control sample, with an ABM of the present invention,under conditions that allow for formation of a complex between the ABMand EGFR. The complex formation is then detected (e.g., by ELISA orother methods known in the art). When using a control sample with thetest sample,any statistically significant difference in the formation ofABM-EGFR complexes when comparing the test and control samples isindicative of the presence of EGFR in the test sample. TABLE 2 SEQ IDCDR Nucleotide Sequence NO Heavy Kabat GACTACAAGATACAC 54 ChainGACTACGCCATCAGC 56 CDR1 GACTACTATATGCAC 58 GACTACAAGATATCC 122 ChothiaGGTTTTACATTCACTGACTAC 60 GGTTACACATTCACTGACTAC 62 GGTTATTCATTCACTGACTAC64 AbM GGTTTTACATTCACTGACTACAAGATACAC 66 GGTTTTACATTGACTGACTACGCCATCAGC68 GGTTTTACATTCACTGACTACTATATGCAC 70 GGTTACACATTCACTGACTACTATATGCAC 72GGTTATTCATTCACTGACTACAAGATACAC 74 GGTTTCACATTCACTGACTACAAGATATCC 124Heavy Kabat TATTTTAATCCTAACAGTGGTTATAGTACCTA 76 ChainCAATGAAAAGTTCAAGAGC CDR2 GGGATCAATCCTAACAGTGGTTATAGTACCTA 78CGCACAGAAGTTCCAGGGC TATTTCAACCCTAACAGCGGTTATAGTACCTA 80CGCACAGAAGTTCCAGGGC TGGATCAATCCTAACAGTGGTTATAGTACCTA 82CGCACAGAAGTTTCAGGGC TGGATCAATCCTAACAGTGGTTATAGTACCTA 84CAGCCCAAGCTTCCAAGGC TGGATCAATCCTAACAGTGGTTATAGTACCTA 86CAACGAGAAGTTCCAAGGC TATTTCAACCCTAACAGCGGTTATTCGAACTA 88CGCACAGAAGTTCCAGGGC TATTTCAACCCTAACAGCGGTTATGCCACGTA 90CGCACAGAAGTTCCAGGGC TACTTCAATCCTAACAGTGGTTATAGTACCTA 126CAGCCCAAGCTTCCAAGGC Chothia AATCCTAACAGTGGTTATAGTACC 92AACCCTAACAGCGGTTATTCGAAC 94 AACCCTAACAGCGGTTATGCCACG 96 AbMTATTTTAATCCTAACAGTGGTTATAGTACC 98 GGGATCAATCCTAACAGTGGTTATAGTACC 100TGGATCAATCCTAACAGTGGTTATAGTACC 102 TATTTCAACCCTAACAGCGGTTATTCGAAC 104TATTTCAACCCTAACAGCGGTTATGCCACG 106 Heavy KabatCTATCCCCAGGCGGTTACTATGTTATGGATGC 108 Chain Chothia C CDR3 AbM

TABLE 3 CDR Amino Acid Sequence SEQ ID NO Heavy Kabat DYKIH 53 ChainDYAIS 55 CDR1 DYYMH 57 DYKIS 123 Chothia GFTFTDY 59 GYTFTDY 61 GYSFTDY63 AbM GFTFTDYKIH 65 GFTFTDYAIS 67 GFTFTDYYMH 69 GYTFTDYYMH 71GYSFTDYKIH 73 GFTFTDYKIS 125 Heavy Kabat YFNPNSGYSTYNEKFKS 75 ChainGINPNSGYSTYAQKFQG 77 CDR2 YFNPNSGYSTYAQKFQG 79 WINPNSGYSTYAQKFQG 81WINPNSGYSTYSPSFQG 83 WINPNSGYSTYNEKFQG 85 YFNPNSGYSNYAQKFQG 87YFNPNSGYATYAQKFQG 89 YFNPNSGYSTYSPSFQG 127 Chothia NPNSGYST 91 NPNSGYSN93 NPNSGYAT 95 AbM YFNPNSGYST 97 GINPNSGYST 99 WINPNSGYST 101 YFNPNSGYSN103 YFNPNSGYAT 105 Heavy Kabat LSPGGYYVMDA 107 Chain Chothia CDR3 AbM

TABLE 4 CDR Amino Acid Sequence SEQ ID NO Kabat Light Chain KASQNINNYLN111 CDR1 RASQGINNYLN 113 Kabat Light Chain NTNNLQT 115 CDR2 Kabat LightChain LQHNSFPT 117 CDR3

TABLE 5 SEQ ID CDR Nucleotide Sequence NO Kabat LightAAAGCAAGTCAGAATATTAACAATTACTTAAAC 112 Chain CDR1CGGGCAAGTCAGGGCATTAACAATTACTTAAAT 114 Kabat Light AATACAAACAATTTGCAAACA116 Chain CDR2 AATACCAACAACTTGCAGACA 118 Kabat LightTTGCAGCATAATAGTTTTCCCACG 119 Chain CDR3

It is known that several mechanism are involved in the therapeuticefficacy of anti-EGFR antibodies, including blocking of ligand (e.g.,EGF, TGF-α, etc.) binding to EGFR and subsequent activation of signalingpathways, antibody dependent cellular cytotoxicity (ADCC), and theinduction of growth arrest or terminal differentiation.

The rat monoclonal antibody ICR62 (IgG2b) was discussed in PCTPublication No. WO 95/20045, which is incorporated herein by referencein its entirety. It was directed to the C epitope of EGFR, and was shownto inhibit ligand binding, inhibit growth in vitro of squamous cellcarcinomas expressing EGFR, and induce regression of xenografts oftumors in athymic mice (WO 95/20045; Modjtahedi et al., Br. J. Cancer73:228-235 (1996)). As a fully rodent antibody, administration of ICR62rat monoclonal antibody to humans resulted a HARA response in somepatients following even a single dose. (WO 95/20045; Modjtahedi et al.,Br. J. Cancer 73:228-235 (1996)).

Chimeric mouse/human antibodies have been described. See, for example,Morrison, S. L. et al., PNAS I1:6851-6854 (November 1984); EuropeanPatent Publication No. 173494; Boulianna, G. L, et al., Nature 312:642(December 1984); Neubeiger, M. S. et al., Nature 314:268 (March 1985);European Patent Publication No. 125023; Tan et al., J. Immunol. 135:8564(November 1985); Sun, L. K et al., Hybridoma 5(1):517 (1986); Sahagan etal., J. Immunol. 137:1066-1074 (1986). See generally, Muron, Nature312:597 (December 1984); Dickson, Genetic Engineering News 5(3) (March1985); Marx, Science 229:455 (August 1985); and Morrison, Science229:1202-1207 (September 1985). IMC-C225 (Erbitux®, Imclone) is achimeric monoclonal antibody directed against EGFR and having a mousevariable region and a human constant region (See Herbst and Shin, Cancer94: 1593-1611 (2002)). The murine portion of IMC-225 is derived fromM225, which was found to bind EGFR and inhibit EGF-induced tyrosinekinase-dependent phosphorylation, as well as inducing apoptosis in tumorcell lines over-expressing EGFR (Herbst and Shin, Cancer 94: 1593-1611(2002)). However, M225 elicited a HAMA reaction in patients treated withthe antibody in Phase I clinical trials (Herbst and Shin, Cancer 94:1593-1611 (2002)). IMC-225 has been tested in vivo and in vitro, and hasbeen used in combination with radiation therapy and chemotherapy in anumber of tumor types, including those associated with poor prognosis(Herbst and Shin, Cancer 94: 1593-1611 (2002)). However, IMC-225 hasbeen associated with toxicities such as allergic and skin reactions inpatients administered the IMC-225 antibody in clincial trials (Herbstand Shin, Cancer 94: 1593-1611 (2002)).

In a particularly preferred embodiment, the chimeric ABM of the presentinvention is a humanized antibody. Methods for humanizing non-humanantibodies are known in the art. For example, humanized ABMs of thepresent invention can be prepared according to the methods of U.S. Pat.No. 5,225,539 to Winter, U.S. Pat. No. 6,180,370 to Queen et al., U.S.Pat. No. 6,632,927 to Adair et al., or U.S. Pat. Appl. Pub. No.2003/0039649 to Foote, the entire contents of each of which is hereinincorporated by reference. Preferably, a humanized antibody has one ormore amino acid residues introduced into it from a source which isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. Humanization can be essentially performed following the methodof Winter and co-workers (Jones et al., Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies. Thesubject humanized anti-EGFR antibodies will comprise constant regions ofhuman immunoglobulin.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method of selecting the human framework sequence is tocompare the sequence of each individual subregion of the full rodentframework (i.e., FR1, FR2, FR3, and FR4) or some combination of theindividual subregions (e.g., FR1 and FR2) against a library of knownhuman variable region sequences that correspond to that frameworksubregion (e.g., as determined by Kabat numbering), and choose the humansequence for each subregion or combination that is the closest to thatof the rodent (Leung, U.S. Patent Application Publication No.2003/0040606A1, published Feb. 27, 2003) (the entire contents of whichare hereby incorporated by reference). Another method uses a particularframework region derived from the consensus sequence of all humanantibodies of a particular subgroup of light or heavy chains. The sameframework may be used for several different humanized antibodies (Carteret al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., JImmunol., 151:2623 (1993)) (the entire contents of each of which areherein incorporated by reference).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models can be generated using computerprograms familiar to those skilled in the art (e.g., InsightII,Accelrys, Inc. (formerly MSI), or at http://swissmodel.expasy.org).These computer programs can illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

In one embodiment, the antibodies of the present invention comprise ahuman Fc region. In a specific embodiment, the human constant region isIgG1, as set forth in SEQ ID NOs 109 and 110, and set forth below: IgG1Nucleotide Sequence (SEQ ID NO:110)ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA IgG1 Amino Acid Sequence (SEQ IDNO:109) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

However, variants and isoforms of the human Fc region are alsoencompassed by the present invention. For example, variant Fc regionssuitable for use in the present invention can be produced according tothe methods taught in U.S. Pat. No. 6,737,056 to Presta (Fc regionvariants with altered effector function due to one or more amino acidmodifications); or in U.S. Pat. Appl. Nos. 60/439,498; 60/456,041;60/514,549; or WO 2004/063351 (variant Fc regions with increased bindingaffinity due to amino acid modification); or in U.S. patent ApplicationSer. No. 10/672,280 or WO 2004/099249 (Fc variants with altered bindingto FcγR due to amino acid modification), the contents of each of whichis herein incorporated by reference in its entirety.

In another embodiment, the antigen binding molecules of the presentinvention are engineered to have enhanced binding affinity according to,for example, the methods disclosed in U.S. Pat. Appl. Pub. No.2004/0132066 to Balint et al., the entire contents of which are herebyincorporated by reference.

In one embodiment, the antigen binding molecule of the present inventionis conjugated to an additional moiety, such as a radiolabel or a toxin.Such conjugated ABMs can be produced by numerous methods that are wellknown in the art.

A variety of radionuclides are applicable to the present invention andthose skilled in the art are credited with the ability to readilydetermine which radionuclide is most appropriate under a variety ofcircumstances. For example, ¹³¹iodine is a well known radionuclide usedfor targeted immunotherapy. However, the clinical usefulness of¹³¹iodine can be limited by several factors including: eight-dayphysical half-life;

dehalogenation of iodinated antibody both in the blood and at tumorsites; and emission characteristics (e.g., large gamma component) whichcan be suboptimal for localized dose deposition in tumor. With theadvent of superior chelating agents, the opportunity for attaching metalchelating groups to proteins has increased the opportunities to utilizeother radionuclides such as ¹¹¹indium and ⁹⁰yttrium. ⁹⁰yttrium providesseveral benefits for utilization in radioimmunotherapeutic applications:the 64 hour half-life of ⁹⁰yttrium is long enough to allow antibodyaccumulation by tumor and, unlike e.g., ¹³¹iodine, ⁹⁰yttrium is a purebeta emitter of high energy with no accompanying gamma irradiation inits decay, with a range in tissue of 100 to 1000 cell diameters.Furthermore, the minimal amount of penetrating radiation allows foroutpatient administration of 90yttrium-labeled antibodies. Additionally,internalization of labeled antibody is not required for cell killing,and the local emission of ionizing radiation should be lethal foradjacent tumor cells lacking the target antigen.

Effective single treatment dosages (i.e., therapeutically effectiveamounts) of ⁹⁰yttrium labeled anti-EGFR antibodies range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of ¹³¹iodinelabeled anti-EGFR antibodies range from between about 5 and about 70mCi, more preferably between about 5 and about 40 mCi. Effective singletreatment ablative dosages (i.e., may require autologous bone marrowtransplantation) of ¹³¹iodine labeled anti-EGFR antibodies range frombetween about 30 and about 600 mCi, more preferably between about 50 andless than about 500 mCi. In conjunction with a chimeric anti-EGFRantibody, owing to the longer circulating half life vis-à-vis murineantibodies, an effective single treatment non-marrow ablative dosages of¹³¹iodine labeled chimeric anti-EGFR antibodies range from between about5 and about 40 mCi, more preferably less than about 30 mCi. Imagingcriteria for, e.g., the ¹¹¹indium label, are typically less than about 5mCi.

With respect to radiolabeled anti-EGFR antibodies, therapy therewith canalso occur using a single therapy treatment or using multipletreatments. Because of the radionuclide component, it is preferred thatprior to treatment, peripheral stem cells (“PSC”) or bone marrow (“BM”)be “harvested” for patients experiencing potentially fatal bone marrowtoxicity resulting from radiation. BM and/or PSC are harvested usingstandard techniques, and then purged and frozen for possible reinfusion.Additionally, it is most preferred that prior to treatment a diagnosticdosimetry study using a diagnostic labeled antibody (e.g., using¹¹¹indium) be conducted on the patient, a purpose of which is to ensurethat the therapeutically labeled antibody (e.g., using ⁹⁰yttrium) willnot become unnecessarily “concentrated” in any normal organ or tissue.

In a preferred embodiment, the present invention is directed to anisolated polynucleotide comprising a sequence that encodes a polypeptidehaving an amino acid sequence in Table 7 below. In a preferredembodiment, the invention is directed to an isolated polynucleotidecomprising a sequence shown in Table 6 below. The invention is furtherdirected to an isolated nucleic acid comprising a sequence at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequenceshown in Table 6 below. In another embodiment, the invention is directedto an isolated nucleic acid comprising a sequence that encodes apolypeptide having an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence in Table 7. Theinvention also encompasses an isolated nucleic acid comprising asequence that encodes a polypeptide having the amino acid sequence ofany of the constructs in Table 7 with conservative amino acidsubstitutions. TABLE 6 SEQ ID CONSTRUCT NUCLEOTIDE SEQUENCE NO ICR62 VHCAGGTCAACCTACTGCAGTCTGGGGCTGCACTGGT 2GAAGCCTGGGGCCTCTGTGAAGTTGTCTTGCAAAG GTTCTGGTTTTACATTCACTGACTACAAGATACACTGGGTGAAGCAGAGTCATGGAAAGAGCCTTGAGT GGATTGGGTATTTTAATCCTAACAGTGGTTATAGTACCTACAATGAAAAGTTCAAGAGCAAGGCCACAT TGACTGCAGACAAATCCACCGATACAGCCTATATGGAGCTTACCAGTCTGACATCTGAGGACTCTGCAAC CTATTACTGTACAAGACTATCCCCAGGGGGTTACTATGTTATGGATGCCTGGGGTCAAGGAGCTTCAGTC ACTGTCTCCTC I-HHACAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 4 AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGATTTACATTCACTGACTACGCCATCAG CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCAATCCTAACAGTGGTTATAG TACCTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACCGCGGACAAATCCACGAGCACAGCCTACAT GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA CTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HHB CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 6AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG GGTTCTGGTTTTACATTCACTGACTACAAGATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 8 AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGGTTCTGGTTTTACATTCACTGACTACAAGATACA CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGATATTTCAACCCTAACAGCGGTTATAG TACCTACAATGAAAAGTTCAAGAGCAGGGTCACCATTACCGCGGACAAATCCACGAGCACAGCCTACAT GGAGCTGAGCAGGCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACTATCCGCAGGCGGTTA CTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLA CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 10AGAAGCCTGGGGCCTCGGTGAAGGTCTCCTGCAA GGCCTCTGGTTTTACATTCACTGACTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGCTGGATCAATCCTAACAGTGGTTATAGTACCTACGCACAGAAGTTTCAGGGCAGGGTCACCA TGACCGCCGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCG TGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCA I-HLBCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 12 AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAAGGGTTCTGGTTTTACATTCACTGACTACAAGATCC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGATACTTCAACCCTAACAGCGGTTATA GTACCTACGCACAGAAGTTCCAGGGCAGGGTCACCATGACCGCCGACACGTCCATCAGCACAGCCTACA TGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTT ACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 14AGAAGCCTGGAGCCTCAGTGAAGGTCTCCTGCAA GGGTTCTGGTTTTACATTCACTGACTACAAGATCCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA GTGGATGGGATACTTCAACCCTAACAGCGGTTACAGTACTTACAACGAGAAGTTCAAGAGCCGGGTCAC CATGACCGCCGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGC CGTGTATTACTGTGCGAGACTATCGCCAGGGGGTTACTATGTTATGGATGCCTGGGGGCAAGGGAGCACC GTGACCGTCTCCTCA I-HHDCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 16AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG GCCTCTGGTTTCACATTCACTGACTACAAGATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHECAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 18AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG GGTTCTGGTTTCACATTCACTGACTACAAGATATCCTGGGTGCGACAGGCTCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHFCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 20AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG GGTTCTGGTTTTACATTCACTGACTACAAGATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATTCGAACTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 22AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG GGTTCTGGTTTTACATTCACTGACTACAAGATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATGCCACGTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA1CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 24 AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAAGGCCTCTGGTTTTACATTCACTGACTACTATATGCA CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGCTGGATCAATCCTAACAGTGGTTATAG TACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC TATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLA2 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 26AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA GGCCTCTGGTTTTACATTCACTGACTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGCTGGATCAATCCTAACAGTGGTTATAGTACCTACAACGAGAAGTTCCAAGGCCAGGTCACC ATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCC ATGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA3CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 28 AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAAGGCCTCTGGTTACACATTCACTGACTACTATATGC ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGCTGGATCAATCCTAACAGTGGTTATA GTACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTATCCCCAGGCGGTTA CTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLA4 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 30AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA GGCCTCTGGTTACACATTCACTGACTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA GTGGATGGGCTGGATCAATCCTAACAGTGGTTATAGTACCTACAACGAGAAGTTCCAAGGCCAGGTCAC CATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGC CATGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACC GTGACCGTCTCCTCA I-HLA5CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 32 AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAAGGCCTCTGGTTTTACATTCACTGACTACTATATGCA CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGCTGGATCAATCCTAACAGTGGTTATAG TACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC TATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLA6 CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 34AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA GGCCTCTGGTTTTACATTCACTGACTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGCTGGATCAATCCTAACAGTGGTTATAGTACCTACAACGAGAAGTTCCAAGGCCAGGTCACC ATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCC ATGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA7CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 36 AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAAGGCCTCTGGTTTTACATTCACTGACTACAAGATCC ACTGGGTGCGACAGGCCCGCGGACAACGGCTCGAGTGGATCGGCTGGATCAATCCTAACAGTGGTTATA GTACCTACAACGAGAAGTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACC TGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTATCCCCAGGCGGTT ACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA I-HLA8 CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 38AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA GGCCTCTGGTTTTACATTCACTGACTACAAGATCCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA GTGGATGGGATATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTCCAGGGCAGGGTCAC CATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGG CCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCAC CGTGACCGTCTCCTCA I-HLA9GAGGTGCAGCTCGTGCAGTCTGGCGCTGAGGTGA 40AGAAGCCTGGCGAGTCGTTGAAGATCTCCTGCAAG GGTTCTGGTTATTCATTCACTGACTACAAGATCCACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG TGGATGGGATATTTCAACCCTAACAGCGGTTATAGTACCTACGCACAGAAGTTCCAGGGCAGGGTCACC ATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA10GAGGTGCAGCTCGTGCAGTCTGGCGCTGAGGTGA 120AGAAGCCTGGCGAGTCGTTGAAGATCTCCTGCAAG GGTTCTGGTTATTCATTCACTGACTACAAGATCCACTGGGTGCGACAGATGCCTGGAAAGGGCCTCGAG TGGATGGGCTACTTCAATCCTAACAGTGGTTATAGTACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCA TCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCA TGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCAG VH SignalATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGC 42 Sequence AGCAGCCACAGGAGCCCACTCCICR62 VL GACATCCAGATGACCCAGTCTCCTTCATTCCTGTCT 44GCATCTGTGGGAGACAGAGTCACTATCAACTGCAA AGCAAGTCAGAATATTAACAATTACTTAAACTGGTATCAGCAAAAGCTTGGAGAAGCTCCCAAACGCCT GATATATAATACAAACAATTTGCAAACAGGCATCCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGAT TACACACTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACATATTTCTGCTTGCAGCATAATAGTTT TCCCACGTTTGGAGCTGGGACCAAGCTGGAACTGAAACGTACG I-KC GATATCCAGATGACCCAGTCTCCATCCTCCCTGTC 46TGCATCTGTCGGAGACCGGGTCACCATCACCTGCC GGGCAAGTCAGGGGCATTAACAATTACTTAAATTGGTACCAGCAGAAGCAGGGAAAGCCCCTAAGCGCC TGATCTATAATACCAACAACTTGCAGACAGGCGTCCCATCAAGGTTCAGCGGCAGTGGATCCGGGACAG AATTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACCTATTACTGCTTGCAGCATAATAG TTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGATCAAGCGTAGG VL Signal ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGCCT 48 SequenceCCTGCTGCTCTGGTTCCCAGGTGCCAGGTGT I-KA GATATCCAGATGACCCAGTCTCCATCCTCCCTGTC50 TGCATCTGTCGGAGACCGGGTCACCATCACCTGCCGGGCAAGTCAGGGCATTAACAATTACTTAAATTGG TACCAGCAGAAGCCAGGGAAAGCCCCTAAGCGCCTGATCTATAATACCAACAACTTGCAGACAGGCGTC CCATCAAGGTTCAGCGGCAGTGGATCCGGGACAGAATACACTCTCACCATCAGCAGCCTGCAGCCTGAA GATTTTGCCACCTATTACTGCTTGCAGCATAATAGTTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGA TCAAGCGTACGGTG I-KBGATATCCAGATGACCCAGTCTCCATCCTCCCTGTC 52TGCATGTCTCGGAGACCGGGTCACCATCACCTGCA AAGCAAGTCAGAATATTAACAATTACTTAAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCTAAGCGCC TGATCTATAATACCAACAACTTGCAGACAGGCGTCCCATCAAGGTTCAGCGGCAGTGGATCCGGGACAG AATACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCCACCTATTACTGCTTGCAGCATAATAG TTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGATCAAGCGTACGGTG

TABLE 7 SEQ ID CONSTRUCT AMINO ACID SEQUENCE NO ICR62 VHQVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWV 1KQSHGKSLEWIGYFNPNSGYSTYNEKFKSKATLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVMDAWG QGASVTVSS I-HHAQVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYAISWV 3RQAPGQGLEWMGGINPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HHBQVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWV 5RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HHCQVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWV 7RQAPGQGLEWMGYFNPNSGYSTYNEKFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLAQVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 9RQAPGQGLEWMGWINPNSGYSTYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLBQVQLVQSGAEVKKPGASVKVSCKGSGFTFTDYKIHWV 11RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLCQVQLVQSGAEVKKPGASVKVSCKGSGFITFTDYKIHW 13VRQAPGQGLEWMGYFNPNSGYSTYNEKFKSRVTMTADTSISTAYMELSRLRSDDTAVYYCARLSPGGYYVMDA WGQGTTVTVSS I-HHDQVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWV 15RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HHEQVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKISWV 17RQAPGQGLEWMGYFNPNSGYSTYAOKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HHFQVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWV 19RQAPGQGLEWMGYFNPNSGYSNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HHGQVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWV 21RQAPGQGLEWMGYFNPNSGYATYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA1QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 23RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA2QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 25RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA3QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWV 27RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA4QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWV 29RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA5QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYYMHWV 31RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA6QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYYMHWV 33RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA7QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYKIHWV 35RQARGQRLEWIGWINPNSGYSTYNEKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA8QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYKIHWV 37RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA9EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYKIHWV 39RQAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWG QGTTVTVSS I-HLA10EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYKIHWV 121RQMPGKGLEWMGYFNPNSGYSTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWG QGTTVTVSS VH SignalMDWTWRILFLVAAATGAHS 41 Sequence ICR62 VLDIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQ 43QKLGEAPKRLIYNTNNLQTGIPSRFSGSGSGTDYTLT ISSLQPEDFATYFCLQHNSFPTFGAGTKLELKRTI-KC DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQ 45QKPGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCLQHNSFPTFGQGTKLEIKRTVL Signal MDMRVPAQLLGLLLLWFPGARC 47 Sequence I-KADIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQ 49QKPGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCLQHNSFPTFGQGTKLEIKRTV I-KBDIQMTQSPSSLSASVGDRVTITCKASQNINNYLNWYQ 51QKPGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCLQHNSFPTFGQGTKLEIKRTV

In another embodiment, the present invention is directed to anexpression vector and/or a host cell which comprise one or more isolatedpolynucleotides of the present invention.

Generally, any type of cultured cell line can be used to express the ABMof the present invention. In a preferred embodiment, HEK293-EBNA cells,CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, othermammalian cells, yeast cells, insect cells, or plant cells are used asthe background cell line to generate the engineered host cells of theinvention.

The therapeutic efficacy of the ABMs of the present invention can beenhanced by producing them in a host cell that further expresses apolynucleotide encoding a polypeptide having GnTIII activity. In apreferred embodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the Golgi localization domain of a Golgi residentpolypeptide. In another preferred embodiment, the expression of the ABMsof the present invention in a host cell that expresses a polynucleotideencoding a polypeptide having GnTIII activity results in ABMs withincreased Fc receptor binding affinity and increased effector function.Accordingly, in one embodiment, the present invention is directed to ahost cell comprising (a) an isolated nucleic acid comprising a sequenceencoding a polypeptide having GnTIII activity; and (b) an isolatedpolynucleotide encoding an ABM of the present invention, such as achimeric, primatized or humanized antibody that binds human EGFR. In apreferred embodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the catalytic domain of GnTIII and the Golgilocalization domain is the localization domain of mannosidase II.Methods for generating such fusion polypeptides and using them toproduce antibodies with increased effector functions are disclosed inU.S. Provisional Pat. Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No.2004/0241817 A1, the entire contents of each of which are expresslyincorporated herein by reference. In another preferred embodiment, thechimeric ABM is a chimeric antibody or a fragment thereof, having thebinding specificity of the rat ICR62 antibody. In a particularlypreferred embodiment, the chimeric antibody comprises a human Fc. Inanother preferred embodiment, the antibody is primatized or humanized.

In one embodiment, one or several polynucleotides encoding an ABM of thepresent invention may be expressed under the control of a constitutivepromoter or, alternately, a regulated expression system. Suitableregulated expression systems include, but are not limited to, atetracycline-regulated expression system, an ecdysone-inducibleexpression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encoding an ABM of thepresent invention are comprised within the host cell system, some ofthem may be expressed under the control of a constitutive promoter,while others are expressed under the control of a regulated promoter.The maximal expression level is considered to be the highest possiblelevel of stable polypeptide expression that does not have a significantadverse effect on cell growth rate, and will be determined using routineexperimentation. Expression levels are determined by methods generallyknown in the art, including Western blot analysis using an antibodyspecific for the ABM or an antibody specific for a peptide tag fused tothe ABM; and Northern blot analysis. In a further alternative, thepolynucleotide may be operatively linked to a reporter gene; theexpression levels of a chimeric (e.g., humanized) ABM havingsubstantially the same binding specificity of the rat ICR62 antibody aredetermined by measuring a signal correlated with the expression level ofthe reporter gene. The reporter gene may be transcribed together withthe nucleic acid(s) encoding said fusion polypeptide as a single mRNAmolecule; their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). The reporter gene may be translated together with atleast one nucleic acid encoding a chimeric (e.g., humanized) ABM havingsubstantially the same binding specificity of the rat ICR62 antibodysuch that a single polypeptide chain is formed. The nucleic acidsencoding the AMBs of the present invention may be operatively linked tothe reporter gene under the control of a single promoter, such that thenucleic acid encoding the fusion polypeptide and the reporter gene aretranscribed into an RNA molecule which is alternatively spliced into twoseparate messenger RNA (mRNA) molecules; one of the resulting mRNAs istranslated into said reporter protein, and the other is translated intosaid fusion polypeptide.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of an ABMhaving substantially the same binding specificity of the rat ICR62antibody along with appropriate transcriptional/translational controlsignals. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Maniatis et al., MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989)and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y (1989).

A variety of host-expression vector systems may be utilized to expressthe coding sequence of the ABMs of the present invention. Preferably,mammalian cells are used as host cell systems transfected withrecombinant plasmid DNA or cosmid DNA expression vectors containing thecoding sequence of the protein of interest and the coding sequence ofthe fusion polypeptide. Most preferably, HEK293-EBNA cells, CHO cells,BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, other mammaliancells, yeast cells, insect cells, or plant cells are used as host cellsystem. Some examples of expression systems and selection methods aredescribed in the following references, and references therein: Borth etal., Biotechnol. Bioen. 71(4):266-73 (2000-2001), in Werner et al.,Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), in Andersen andKrummen, Curr. Op. Biotechnol. 13:117-123 (2002), in Chadd and Chamow,Curr. Op. Biotechnol. 12:188-194 (2001), and in Giddings, Curr. Op.Biotechnol. 12: 450-454 (2001). In alternate embodiments, othereukaryotic host cell systems may be used, including yeast cellstransformed with recombinant yeast expression vectors containing thecoding sequence of an ABM of the present invention, such as theexpression systems taught in U.S. Pat. Appl. No. 60/344,169 and WO03/056914 (methods for producing human-like glycoprotein in a non-humaneukaryotic host cell) (the contents of each of which are incorporated byreference in their entirety); insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing thecoding sequence of a chimeric ABM having substantially the same bindingspecificity of the rat ICR62 antibody; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing the coding sequence ofthe ABM of the invention, including, but not limited to, the expressionsystems taught in U.S. Pat. No. 6,815,184 (methods for expression andsecretion of biologically active polypeptides from geneticallyengineered duckweed); WO 2004/057002 (production of glycosylatedproteins in bryophyte plant cells by introduction of a glycosyltransferase gene) and WO 2004/024927 (methods of generatingextracellular heterologous non-plant protein in moss protoplast); andU.S. Pat. Appl. Nos. 60/365,769, 60/368,047, and WO 2003/078614(glycoprotein processing in transgenic plants comprising a functionalmammalian GnTIII enzyme) (the contents of each of which are hereinincorporated by reference in its entirety); or animal cell systemsinfected with recombinant virus expression vectors (e.g., adenovirus,vaccinia virus) including cell lines engineered to contain multiplecopies of the DNA encoding a chimeric ABM having substantially the samebinding specificity of the rat ICR62 antibody either stably amplified(CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g.,murine cell lines). In one embodiment, the vector comprising thepolynucleotide(s) encoding the ABM of the invention is polycistronic.Also, in one embodiment the ABM discussed above is an antibody or afragment thereof. In a preferred embodiment, the ABM is a humanizedantibody.

For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large-scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes, which can be employed in tk-, hgprt- or aprt-cells, respectively.Also, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler et al., Natl.Acad. Sci. USA 77:3567 (1989); O'Hare et al., Proc. Natl. Acad. Sci. USA78:1527 (1981)); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,J. Mol. Biol. 150:1 (1981)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)); the glutaminesynthase system; and ODC (omithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-omithine, DFMO (McConlogue, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.(1987)).

The present invention is further directed to a method for modifying theglycosylation profile of the ABMs of the present invention that areproduced by a host cell, comprising expressing in said host cell anucleic acid encoding an ABM of the invention and a nucleic acidencoding a polypeptide with GnTIII activity, or a vector comprising suchnucleic acids. Preferably, the modified polypeptide is IgG or a fragmentthereof comprising the Fc region. In a particularly preferred embodimentthe ABM is a humanized antibody or a fragment thereof.

The modified ABMs produced by the host cells of the invention exhibitincreased Fc receptor binding affinity and/or increased effectorfunction as a result of the modification. In a particularly preferredembodiment the ABM is a humanized antibody or a fragment thereofcontaining the Fc region. Preferably, the increased Fc receptor bindingaffinity is increased binding to a Fcγ activating receptor, such as theFcγRIIIa receptor. The increased effector function is preferably anincrease in one or more of the following: increased antibody-dependentcellular cytotoxicity, increased antibody-dependent cellularphagocytosis (ADCP), increased cytokine secretion, increasedimmune-complex-mediated antigen uptake by antigen-presenting cells,increased Fc-mediated cellular cytotoxicity, increased binding to NKcells, increased binding to macrophages, increased binding topolymorphonuclear cells (PMNs), increased binding to monocytes,increased crosslinking of target-bound antibodies, increased directsignaling inducing apoptosis, increased dendritic cell maturation, andincreased T cell priming.

The present invention is also directed to a method for producing an ABMof the present invention, having modified oligosaccharides in a hostcell comprising (a) culturing a host cell engineered to express at leastone nucleic acid encoding a polypeptide having GnTIll activity underconditions which permit the production of an ABM according to thepresent invention, wherein said polypeptide having GnTIII activity isexpressed in an amount sufficient to modify the oligosaccharides in theFc region of said ABM produced by said host cell; and (b) isolating saidABM. In a preferred embodiment, the polypeptide having GnTIII activityis a fusion polypeptide comprising the catalytic domain of GnTIII. In aparticularly preferred embodiment, the fusion polypeptide furthercomprises the Golgi localization domain of a Golgi resident polypeptide.

Preferably, the Golgi localization domain is the localization domain ofmannosidase II or GnTI. Alternatively, the Golgi localization domain isselected from the group consisting of: the localization domain ofmannosidase I, the localization domain of GnTII, and the localizationdomain of α 1-6 core fucosyltransferase. The ABMs produced by themethods of the present invention have increased Fc receptor bindingaffinity and/or increased effector function. Preferably, the increasedeffector function is one or more of the following: increased Fc-mediatedcellular cytotoxicity (including increased antibody-dependent cellularcytotoxicity), increased antibody-dependent cellular phagocytosis(ADCP), increased cytokine secretion, increased immune-complex-mediatedantigen uptake by antigen-presenting cells, increased binding to NKcells, increased binding to macrophages, increased binding to monocytes,increased binding to polymorphonuclear cells, increased direct signalinginducing apoptosis, increased crosslinking of target-bound antibodies,increased dendritic cell maturation, or increased T cell priming. Theincreased Fc receptor binding affinity is preferably increased bindingto Fc activating receptors such as FcγRIIIa. In a particularly preferredembodiment the ABM is a humanized antibody or a fragment thereof.

In another embodiment, the present invention is directed to a chimericABM having substantially the same binding specificity of the rat ICR62antibody produced by the methods of the invention which has an increasedproportion of bisected oligosaccharides in the Fc region of saidpolypeptide. It is contemplated that such an ABM encompasses antibodiesand fragments thereof comprising the Fc region. In a preferredembodiment, the ABM is a humanized antibody. In one embodiment, thepercentage of bisected oligosaccharides in the Fc region of the ABM isat least 50%, more preferably, at least 60%, at least 70%, at least 80%,or at least 90%, and most preferably at least 90-95% of the totaloligosaccharides. In yet another embodiment, the ABM produced by themethods of the invention has an increased proportion of nonfucosylatedoligosaccharides in the Fc region as a result of the modification of itsoligosaccharides by the methods of the present invention. In oneembodiment, the percentage of nonfucosylated oligosaccharides is atleast 50%, preferably, at least 60% to 70%, most preferably at least75%. The nonfucosylated oligosaccharides may be of the hybrid or complextype. In a particularly preferred embodiment, the ABM produced by thehost cells and methods of the invention has an increased proportion ofbisected, nonfucosylated oligosaccharides in the Fc region. Thebisected, nonfucosylated oligosaccharides may be either hybrid orcomplex. Specifically, the methods of the present invention may be usedto produce ABMs in which at least 15%, more preferably at least 20%,more preferably at least 25%, more preferably at least 30%, morepreferably at least 35% of the oligosaccharides in the Fc region of theABM are bisected, nonfucosylated. The methods of the present inventionmay also be used to produce polypeptides in which at least 15%, morepreferably at least 20%, more preferably at least 25%, more preferablyat least 30%, more preferably at least 35% of the oligosaccharides inthe Fc region of the polypeptide are bisected hybrid nonfucosylated.

In another embodiment, the present invention is directed to a chimericABM having substantially the same binding specificity of the rat ICR62antibody engineered to have increased effector function and/or increasedFc receptor binding affinity, produced by the methods of the invention.Preferably, the increased effector function is one or more of thefollowing: increased Fc-mediated cellular cytotoxicity (includingincreased antibody-dependent cellular cytotoxicity), increasedantibody-dependent cellular phagocytosis (ADCP), increased cytokinesecretion, increased immune-complex-mediated antigen uptake byantigen-presenting cells, increased binding to NK cells, increasedbinding to macrophages, increased binding to monocytes, increasedbinding to polymorphonuclear cells, increased direct signaling inducingapoptosis, increased crosslinking of target-bound antibodies, increaseddendritic cell maturation, or increased T cell priming. In a preferredembodiment, the increased Fc receptor binding affinity is increasedbinding to a Fc activating receptor, most preferably FcγRIIIa. In oneembodiment, the ABM is an antibody, an antibody fragment containing theFc region, or a fusion protein that includes a region equivalent to theFc region of an immunoglobulin. In a particularly preferred embodiment,the ABM is a humanized antibody.

The present invention is further directed to pharmaceutical compositionscomprising the ABMs of the present invention and a pharmaceuticallyacceptable carrier.

The present invention is further directed to the use of suchpharmaceutical compositions in the method of treatment of cancer.Specifically, the present invention is directed to a method for thetreatment of cancer comprising administering a therapeutically effectiveamount of the pharmaceutical composition of the invention.

The present invention further provides methods for the generation anduse of host cell systems for the production of glycoforms of the ABMs ofthe present invention, having increased Fc receptor binding affinity,preferably increased binding to Fc activating receptors, and/or havingincreased effector functions, including antibody-dependent cellularcytotoxicity. The glycoengineering methodology that can be used with theABMs of the present invention has been described in greater detail inU.S. Pat. No. 6,602,684, U.S. Pat. Appl. Publ. No. 2004/0241817 A1, U.S.Pat. Appl. Publ. No. 2003/0175884 A1, Provisional U.S. PatentApplication No. 60/441,307 and WO 2004/065540, the entire contents ofeach of which is incorporated herein by reference in its entirety. TheABMs of the present invention can alternatively be glycoengineered tohave reduced fucose residues in the Fc region according to thetechniques. disclosed in U.S. Pat. Appl. Pub. No. 2003/0157108(Genentech), or in EP 1 176 195 A1, WO 03/084570, WO 03/085119 and U.S.Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621, 2004/110282,2004/110704, 2004/132140 (Kyowa). The contents of each of thesedocuments are herein incorporated by reference in their entireties.Glycoengineered ABMs of the invention may also be produced in expressionsystems that produce modified glycoproteins, such as those taught inU.S. Pat. Appl. Pub. No. 60/344,169 and WO 03/056914 (GlycoFi, Inc.) orin WO 2004/057002 and WO 2004/024927 (Greenovation), the contents ofeach of which are hereby incorporated by reference in their entirety.

Generation of Cell Lines for the Production of Proteins with AlteredGlycosylation Pattern

The present invention provides host cell expression systems for thegeneration of the ABMs of the present invention having modifiedglycosylation patterns. In particular, the present invention provideshost cell systems for the generation of glycoforms of the ABMs of thepresent invention having an improved therapeutic value. Therefore, theinvention provides host cell expression systems selected or engineeredto express a polypeptide having GnTIII activity. In one embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide comprisingthe Golgi localization domain of a heterologous Golgi residentpolypeptide. Specifically, such host cell expression systems may beengineered to comprise a recombinant nucleic acid molecule encoding apolypeptide having GnTIII, operatively linked to a constitutive orregulated promoter system.

In one specific embodiment, the present invention provides a host cellthat has been engineered to express at least one nucleic acid encoding afusion polypeptide having GnTIII activity and comprising the Golgilocalization domain of a heterologous Golgi resident polypeptide. In oneaspect, the host cell is engineered with a nucleic acid moleculecomprising at least one gene encoding a fusion polypeptide having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide.

Generally, any type of cultured cell line, including the cell linesdiscussed above, can be used as a background to engineer the host celllines of the present invention. In a preferred embodiment, HEK293-EBNAcells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells,P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells,other mammalian cells, yeast cells, insect cells, or plant cells areused as the background cell line to generate the engineered host cellsof the invention.

The invention is contemplated to encompass any engineered host cellsexpressing a polypeptide having GnTIII activity, including a fusionpolypeptide that comprises the Golgi localization domain of aheterologous Golgi resident polypeptide as defined herein.

One or several nucleic acids encoding a polypeptide having GnTIIIactivity may be expressed under the control of a coristitutive promoteror, alternately, a regulated expression system. Such systems are wellknown in the art, and include the systems discussed above. If severaldifferent nucleic acids encoding fusion polypeptides having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide are comprised within the host cell system,some of them may be expressed under the control of a constitutivepromoter, while others are expressed under the control of a regulatedpromoter. Expression levels of the fusion polypeptides having GnTIIIactivity are determined by methods generally known in the art, includingWestern blot analysis, Northern blot analysis, reporter gene expressionanalysis or measurement of GnTIII activity. Alternatively, a lectin maybe employed which binds to biosynthetic products of the GnTIII, forexample, E₄-PHA lectin. Alternatively, a functional assay which measuresthe increased Fc receptor binding or increased effector functionmediated by antibodies produced by the cells engineered with the nucleicacid encoding a polypeptide with GnTIII activity may be used.

Identification of Transfectants or Transformants that Express theProtein Having A Modified Glycosylation Pattern

The host cells which contain the coding sequence of a chimeric (e.g.,humanized) ABM having substantially the same binding specificity of therat ICR62 antibody and which express the biologically active geneproducts may be identified by at least four general approaches; (a)DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of“marker” gene functions; (c) assessing the level of transcription asmeasured by the expression of the respective MRNA transcripts in thehost cell; and (d) detection of the gene product as measured byimmunoassay or by its biological activity.

In the first approach, the presence of the coding sequence of a chimeric(e.g., humanized) ABM having substantially the same binding specificityof the rat ICR62 antibody and the coding sequence of the polypeptidehaving GnTIII activity can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the respective coding sequences, respectively, or portionsor derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.).

For example, if the coding sequence of the ABM of the invention, or afragment thereof, and the coding sequence of the polypeptide havingGnTIII activity are inserted within a marker gene sequence of thevector, recombinants containing the respective coding sequences can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the coding sequences under thecontrol of the same or different promoter used to control the expressionof the coding sequences. Expression of the marker in response toinduction or selection indicates expression of the coding sequence ofthe ABM of the invention and the coding sequence of the polypeptidehaving GnTIII activity.

In the third approach, transcriptional activity for the coding region ofthe ABM of the invention, or a fragment thereof, and the coding sequenceof the polypeptide having GnTIII activity can be assessed byhybridization assays. For example, RNA can be isolated and analyzed byNorthern blot using a probe homologous to the coding sequences of theABM of the invention, or a fragment thereof, and the coding sequence ofthe polypeptide having GnTIII activity or particular portions thereof.Alternatively, total nucleic acids of the host cell may be extracted andassayed for hybridization to such probes.

In the fourth approach, the expression of the protein products can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active geneproducts.

Generation and Use of ABMs Having Increased Effector Function IncludingAntibody-Dependent Cellular Cytotoxicity

In preferred embodiments, the present invention provides glycoforms ofchimeric (e.g., humanized) ABMs having substantially the same bindingspecificity of the rat ICR62 antibody and having increased effectorfunction including antibody-dependent cellular cytotoxicity.Glycosylation engineering of antibodies has been previously described.See e.g., U.S. Pat. No. 6,602,684, incorporated herein by reference inits entirety.

Clinical trials of unconjugated monoclonal antibodies (mAbs) for thetreatment of some types of cancer have recently yielded encouragingresults. Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); Deo etal., Immunology Today 18:127 (1997). A chimeric, unconjugated IgG1 hasbeen approved for low-grade or follicular B-cell non-Hodgkin's lymphoma.Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997), while anotherunconjugated mAb, a humanized IgG1 targeting solid breast tumors, hasalso been showing promising results in phase III clinical trials. Deo etal., Immunology Today 18:127 (1997). The antigens of these two mAbs arehighly expressed in their respective tumor cells and the antibodiesmediate potent tumor destruction by effector cells in vitro and in vivo.In contrast, many other unconjugated mAbs with fine tumor specificitiescannot trigger effector functions of sufficient potency to be clinicallyuseful. Frost et al., Cancer 80:317-33 (1997); Surfus et al., J.Immunother. 19:184-91 (1996). For some of these weaker mAbs, adjunctcytokine therapy is currently being tested. Addition of cytokines canstimulate antibody-dependent cellular cytotoxicity (ADCC) by increasingthe activity and number of circulating lymphocytes. Frost et al., Cancer80:317-33 (1997); Surfus et al., J. Immunother. 19:184-91 (1996). ADCC,a lytic attack on antibody-targeted cells, is triggered upon binding ofleukocyte receptors to the constant region (Fc) of antibodies. Deo etal., Immunology Today 18:127 (1997).

A different, but complementary, approach to increase ADCC activity ofunconjugated IgG1s is to engineer the Fc region of the antibody. Proteinengineering studies have shown that FcγRs interact with the lower hingeregion of the IgG CH2 domain. Lund et al., J. Immunol. 157:4963-69(1996). However, FcγR binding also requires the presence ofoligosaccharides covalently attached at the conserved Asn 297 in the CH2region. Lund et al., J. Immunol. 157:4963-69 (1996); Wright andMorrison, Trends Biotech. 15:26-31 (1997), suggesting that eitheroligosaccharide and polypeptide both directly contribute to theinteraction site or that the oligosaccharide is required to maintain anactive CH2 polypeptide conformation. Modification of the oligosaccharidestructure can therefore be explored as a means to increase the affinityof the interaction.

An IgG molecule carries two N-linked oligosaccharides in its Fc region,one on each heavy chain. As any glycoprotein, an antibody is produced asa population of glycoforms which share the same polypeptide backbone buthave different oligosaccharides attached to the glycosylation sites. Theoligosaccharides normally found in the Fc region of serum IgG are ofcomplex bi-antennary type (Wormald et al., Biochemistry 36:130-38(1997), with a low level of terminal sialic acid and bisectingN-acetylglucosamine (GlcNAc), and a variable degree of terminalgalactosylation and core fucosylation. Some studies suggest that theminimal carbohydrate structure required for FcγR binding lies within theoligosaccharide core. Lund et al., J. Immunol. 157:4963-69 (1996).

The mouse- or hamster-derived cell lines used in industry and academiafor production of unconjugated therapeutic mAbs normally attach therequired oligosaccharide determinants to Fc sites. IgGs expressed inthese cell lines lack, however, the bisecting GlcNAc found in lowamounts in serum IgGs. Lifely et al., Glycobiology 318:813-22 (1995). Incontrast, it was recently observed that a rat myeloma-produced,humanized IgG1 (CAMPATH-1H) carried a bisecting GlcNAc in some of itsglycoforms. Lifely et al., Glycobiology 318:813-22 (1995). The ratcell-derived antibody reached a similar maximal in vitro ADCC activityas CAMPATH-1H antibodies produced in standard cell lines, but atsignificantly lower antibody concentrations.

The CAMPATH antigen is normally present at high levels on lymphomacells, and this chimeric mAb has high ADCC activity in the absence of abisecting GlcNAc.

Lifely et al., Glycobiology 318:813-22 (1995). In the N-linkedglycosylation pathway, a bisecting GlcNAc is added by GnTIII. Schachter,Biochem. Cell Biol. 64:163-81 (1986).

Previous studies used a single antibody-producing CHO cell line, thatwas previously engineered to express, in an externally-regulatedfashion, different levels of a cloned GnT III gene enzyme (Umana, P., etal., Nature Biotechnol. 17:176-180 (1999)). This approach establishedfor the first time a rigorous correlation between expression of GnTIIIand the ADCC activity of the modified antibody. Thus, the inventioncontemplates a recombinant, chimeric antibody or a fragment thereof withthe binding specificity of the rat ICR62 antibody, having alteredglycosylation resulting from increased GnTIII activity. The increasedGnTIII activity results in an increase in the percentage of bisectedoligosaccharides, as well as a decrease in the percentage of fucoseresidues, in the Fc region of the ABM. This antibody, or fragmentthereof, has increased Fc receptor binding affinity and increasedeffector function. In addition, the invention is directed to antibodyfragment and fusion proteins comprising a region that is equivalent tothe Fc region of immunoglobulins. In a preferred embodiment, theantibody is humanized.

Therapeutic Applications of ABMs Produced According to the Methods ofthe Invention.

In the broadest sense, the ABMs of the present invention can be usedtarget cells in vivo or in vitro that express EGFR. The cells expressingEGFR can be targetted for diagnostic or therapeutic purposes. In oneaspect, the ABMs of the present invention can be used to detect thepresence of EGFR in a sample. In another aspect, the ABMs of the presentinvention can be used to inhibit or reduce EGFR-mediated signaltransduction in cells expressing EGFR on the surface. EGFR is abnormallyexpressed (e.g., overexpressed) in many human tumors compared tonon-tumor tissue of the same cell type. Thus, the ABMs of the inventionare particularly useful in the prevention of tumor formation,eradication of tumors and inhibition of tumor growth. By blocking thebinding of EGFR ligands to EGFR, the ABMs of the invention inhibitEGF-dependent tumor cell activation, including EGFR tyrosinephosphorylation, increased extracellular acidification rate, and cellproliferation. The ABMs of the invention also act to arrest the cellcycle, cause apoptosis of the target cells (e.g., tumor cells), andinhibit angiogenesis and/or differentiation of target cells. The ABMs ofthe invention can be used to treat any tumor expressing EGFR. Particularmalignancies that can be treated with the ABMs of the invention include,but are not limited to, epidermal and squamous cell carcinomas,non-small cell lung carcinomas, gliomas, pancreatic cancer, ovariancancer, prostate cancer, breast cancer, bladder cancer, head and neckcancer, and renal cell carcinomas.

The ABMs of the present can be used alone to target and kill tumor cellsin vivo. The ABMs can also be used in conjunction with an appropriatetherapeutic agent to treat human carcinoma. For example, the ABMs can beused in combination with standard or conventional treatment methods suchas chemotherapy, radiation therapy or can be conjugated or linked to atherapeutic drug, or toxin, as well as to a lymphokine or atumor-inhibitory growth factor, for delivery of the therapeutic agent tothe site of the carcinoma. The conjugates of the ABMs of this inventionthat are of prime importance are (1) immunotoxins (conjugates of the ABMand a cytotoxic moiety) and (2) labeled (e.g. radiolabeled,enzyme-labeled, or fluorochrome-labeled) ABMs in which the labelprovides a means for identifying immune complexes that include thelabeled ABM. The ABMs can also be used to induce lysis through thenatural complement process, and to interact with antibody dependentcytotoxic cells normally present.

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or anenzymatically active toxin of bacterial or plant origin, or anenzymatically active fragment (“A chain”) of such a toxin. Enzymaticallyactive toxins and fragments thereof used are diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaccaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, and enomycin. In anotherembodiment, the ABMs are conjugated to small molecule anticancer drugs.

Conjugates of the ABM and such cytotoxic moieties are made using avariety of bifunctional protein coupling agents. Examples of suchreagents are SPDP, IT, bi functional derivatives of imidoesters such adimethyl adipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such asbis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyanate, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may bejoined to the Fab fragment of the ABMs. Additional appropriate toxinsare known in the art, as evidenced in e.g., published U.S. PatentApplication No. 2002/0128448, incorporated herein by reference in itsentirety.

In one embodiment, a chimeric (e.g., humanized), glycoengineered ABMhaving substantially the same binding specificity of the rat ICR62antibody, is conjugated to ricin A chain. Most advantageously, the ricinA chain is deglycosylated and produced through recombinant means. Anadvantageous method of making the ricin immunotoxin is described inVitetta et al., Science 238, 1098 (1987), hereby incorporated byreference.

When used to kill human cancer cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques todetermine the presence or degree of cancer.

As discussed above, a cytotoxic radiopharmaceutical for treating cancermay be made by conjugating a radioactive isotope (e.g., I, Y, Pr) to achimeric, glycoengineered ABM having substantially the same bindingspecificity of the rat ICR62 antibody. The term “cytotoxic moiety” asused herein is intended to include such isotopes.

In another embodiment, liposomes are filled with a cytotoxic drug andthe liposomes are coated with the ABMs of the present invention. Becausethere are many EGFR molecules on the surface of the EGFR-expressingmalignant cell, this method permits delivery of large amounts of drug tothe correct cell type.

Techniques for conjugating such therapeutic agents to antibodies arewell known (see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)).

Still other therapeutic applications for the ABMs of the inventioninclude conjugation or linkage, e.g., by recombinant DNA techniques, toan enzyme capable of converting a prodrug into a cytotoxic drug and theuse of that antibody-enzyme conjugate in combination with the prodrug toconvert the prodrug to a cytotoxic agent at the tumor site (see, e.g.,Senter et al., “Anti-Tumor Effects of Antibody-alkaline Phosphatase”,Proc. Natl. Acad. Sci. USA 85:4842-46 (1988); “Enhancement of the invitro and in vivo Antitumor Activites of Phosphorylated Mitocycin C andEtoposide Derivatives by Monoclonal Antibody-Alkaline PhosphataseConjugates”, Cancer Research 49:5789-5792 (1989); and Senter,“Activation of Prodrugs by Antibody-Enzyme Conjugates: A New Approach toCancer Therapy,” FASEB J. 4:188-193 (1990)).

Still another therapeutic use for the ABMs of the invention involvesuse, either unconjugated, in the presence of complement, or as part ofan antibody-drug or antibody-toxin conjugate, to remove tumor cells fromthe bone marrow of cancer patients. According to this approach,autologous bone marrow may be purged ex vivo by treatment with theantibody and the marrow infused back into the patient [see, e.g., Ramsayet al., “Bone Marrow Purging Using Monoclonal Antibodies”, J. Clin.Immunol., 8(2):81-88 (1988)].

Furthermore, it is contemplated that the invention comprises asingle-chain immunotoxin comprising antigen binding domains that allowsubstantially the same specificity of binding as the rat ICR62 antibody(e.g., polypeptides comprising the CDRs of the rat ICR62 antibody) andfurther comprising a toxin polypeptide. The single-chain immunotoxins ofthe invention may be used to treat human carcinoma in vivo.

Similarly, a fusion protein comprising at least the antigen-bindingregion of an ABM of the invention joined to at least a functionallyactive portion of a second protein having anti-tumor acitivty, e.g., alymphokine or oncostatin, can be used to treat human carcinoma in vivo.

The present invention provides a method for selectively killing tumorcells expressing EGFR. This method comprises reacting theimmunoconjugate (e.g., the immunotoxin) of the invention with said tumorcells. These tumor cells may be from a human carcinoma.

Additionally, this invention provides a method of treating carcinomas(for example, human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g., theimmunotoxin) of the invention.

In a further aspect, the invention is directed to an improved method fortreating cell proliferation disorders wherein EGFR is expressed,particularly wherein EGFR is abnormally expressed (e.g. overexpressed),including cancers of the bladder, brain, head and neck, pancreas, lung,breast, ovary, colon, prostate, skin, and kidney, comprisingadministering a therapeutically effective amount of an ABM of thepresent invention to a human subject in need thereof. In a preferredembodiment, the ABM is a glycoengineered anti-EGFR antibody with abinding specificity substantially the same as that of the rat ICR62antibody. In another preferred embodiment the antibody is humanized.Examples of cell proliferation disorders that can be treated by an ABMof the present invention include, but are not limited to neoplasmslocated in the: abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system.

Similarly, other cell proliferation disorders can also be treated by theABMs of the present invention. Examples of such cell proliferationdisorders include, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other cell proliferation disease, besidesneoplasia, located in an organ system listed above.

In accordance with the practice of this invention, the subject may be ahuman, equine, porcine, bovine, murine, canine, feline, and aviansubjects. Other warm blooded animals are also included in thisinvention.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

The invention is further directed to methods for treating non-malignantdiseases or disorders in a mammal characterized by abnormal activationor production of EGFR or one or more EGFR ligands, comprisingadministering to the mammal a therapeutically effective amount of theABMs of the invention. The subject will generally have EGFR-expressingcells, for instance in diseased tissue thereof, such that the ABMs ofthe invention are able to bind to cells within the subject.

Abnormal activation or expression of EGFR or an EGFR ligand may beoccurring in cells of the subject, e.g. in diseased tissue of thesubject. Abnormal activation of EGFR may be attributable toamplification, overexpression or aberrant production of the EGFR and/orEGFR ligand. In one embodiment of the invention, a diagnostic orprognostic assay will be performed to determine whether abnormalproduction or activation of EGFR (or EGFR ligand) is occurring thesubject. For example, gene amplification and/or overexpression of EGFRand/or ligand may be determined. Various assays for determining suchamplification/overexpression are available in the art and include theIHC, FISH and shed antigen assays described above. Alternatively, oradditionally, levels of an EGFR ligand, such as TGF-α in or associatedwith the sample may be determined according to known procedures. Suchassays may detect protein and/or nucleic acid encoding it in the sampleto be tested. In one embodiment, EGFR ligand levels in a sample may bedetermined using immunohistochemistry (IHC); see, for example, Scher etal. Clin. Cancer Research 1:545-550 (1995). Alternatively, oradditionally, one may evaluate levels of EGFR -encoding nucleic acid inthe sample to be tested; e.g. via FISH, southern blotting, or PCRtechniques.

Moreover, EGFR or EGFR ligand overexpression or amplification may beevaluated using an in vivo diagnostic assay, e.g. by administering amolecule (such as an antibody) which binds the molecule to be detectedand is tagged with a detectable label (e.g. a radioactive isotope) andexternally scanning the patient for localization of the label.

Alternatively, one may detect EGFR heterodimers, especially EGFR-ErbB2,EGFR -ErbB3 or EGFR -ErbB4 heterodimers, in the patient, e.g. indiseased tissue thereof, prior to therapy. Various methods to detectnoncovalent protein-protein interactions or otherwise indicate proximitybetween proteins of interest are available.

Exemplary methods for detecting EGFR heterodimers include, withoutlimitation, immunoaffinity-based methods, such as immunoprecipitation;fluorescence resonance energy transfer (FRET) (Selvin, Nat. Struct.Biol. 7:730-34 (2000); Gadella & Jovin, J. Cell Biol. 129:1543-58(1995); and Nagy et al., Cytometry 32:120-131 (1998)); co-localizationof EGFR with either ErbB2 or ErbB3 using standard direct or indirectimmunofluorescence techniques and confocal laser scanning microscopy;laser scanning imaging (LSI) to detect antibody binding andco-localization of EGFR with either ErbB2 or ErbB3 in a high-throughputformat, such as a microwell plate (Zuck et al, Proc. Natl. Acad. Sci.USA 96:11122-11127 (1999)); or eTag/m assay system (Aclara Bio Sciences,Mountain View, Calif.; and U.S. Patent Application 2001/0049105,published Dec. 6, 2001).

It is apparent, therefore, that the present invention encompassespharmaceutical compositions, combinations and methods for treating humanmalignancies such as cancers of the bladder, brain, head and neck,pancreas, lung, breast, ovary, colon, prostate, skin, and kidney. Forexample, the invention includes pharmaceutical compositions for use inthe treatment of human malignancies comprising a pharmaceuticallyeffective amount of an antibody of the present invention and apharmaceutically acceptable carrier.

The ABM compositions of the invention can be administered usingconventional modes of administration including, but not limited to,intravenous, intraperitoneal, oral, intralymphatic or administrationdirectly into the tumor. Intravenous administration is preferred.

In one aspect of the invention, therapeutic formulations containing theABMs of the invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (REMINGTON'SPHARMACEUTICAL SCIENCES, 16^(th) edition, Osol, A. Ed. (1980)), in theform of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The ABMs of the present invention may be administered to a subject totreat a disease or disorder characterized by abnormal EGFR or EGFRligand activity, such as a tumor, either alone or in combination therapywith, for example, a chemotherapeutic agent and/or radiation therapy.Exemplary anti-EGFR antibody formulations are described in Herbst andShen, Cancer 94(5):1593-161 1. Suitable chemotherapeutic agents includecisplatin, doxorubicin, topotecan, paclitaxel, vinblastine, carboplatin,and etoposide.

Lyophilized formulations adapted for subcutaneous administration aredescribed in WO97/04801. Such lyophihized formulations may bereconstituted with a suitable diluent to a high protein concentrationand the reconstituted formulation may be administered subcutaneously tothe mammal to be treated herein.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide a cytotoxic agent,chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. onewhich acts on T cells, such as cyclosporin or an antibody that binds Tcells, e.g., one which binds LFA-1). The effective amount of such otheragents depends on the amount of antagonist present in the formulation,the type of disease or disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(yinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspension,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thepharmaceutical compositions of this invention depends upon the severityand course of the disease, the patient's health and response totreatment and the judgment of the treating physician. Accordingly, thedosages of the compositions should be titrated to the individualpatient. Nevertheless, an effective dose of the compositions of thisinvention will generally be in the range of from about 0.01 to about2000 mg/kg. In one embodiment, the effective dose is in the range offrom about 1.0 mg/kg to about 15.0 mg/kg. In a more specific embodiment,the dose is in the range of from about 1.5 mg/kg to about 12 mg/kg. Inother embodiments, the dose is in the range of from about 1.5 mg/kg toabout 4.5 mg/kg, or from about 4.5 mg/kg to about 12 mg/kg. The dose ofthe present invention may also be any dose within these ranges,including, but not limited to, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg,6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0 mg/kg, 11.5 mg/kg, 12.0mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0 mg/kg, 14.5 mg/kg, or15.0 mg/kg.

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The dosages of the present invention may, in some cases, be determinedby the use of predictive biomarkers. Predictive biomarkers are molecularmarkers that are used to determine (i.e., observe and/or quanitate) apattern of expression and/or activation of tumor related genes orproteins, or cellular components of a tumor related signalling pathway.Elucidating the biological effects of targeted-therapies in tumor tissueand correlating these effects with clinical response helps identify thepredominant growth and survival pathways operative in tumors, therebyestablishing a profile of likely responders and conversely providing arational for designing strategies to overcoming resistance. For example,biomarkers for anti-EGFR therapy may comprise molecules that are in theEGFR downstream signalling pathway that leads to a cell proliferationdisorder including, but not limited to, Akt, RAS, RAF, MAPK, ERKI, ERK2,PKC, STAT3, STAT5 (Mitchell, Nature Biotech. 22: 363-364 (2004); Becker,Nature Biotech 22:15-18 (2004); Tsao and Herbst, Signal 4:4-9 (2003)).Biomarkers for anti-EGFR therapy may also comprise growth factorreceptors such as EGFR, ErbB-2 (HER2/neu), and ErbB-3 (HER3), and may bepositive or negative predictors of patient response to anti-EGFRtherapy. For example, the growth factor receptor ErbB-3 (HER3) wasdetermined to be a negative predictive biomarker for the anti-EGFRantibody ABX-EGF (U.S. Pat. Appl. Pub. No. 2004/0132097 A1).

Predictive biomarkers may be measured by cellular assays that are wellknown in the art including, but not limited to immunohistochemistry,flow cytometry, immunofluorescence, capture-and-detection assays, andreversed phase assays, and/or assays set forth in U.S. Pat. Appl. Pub.No. 2004/0132097 A1, the entire contents of which are hereinincorporated by reference . Predictive biomarkers of anti-EGFR therapy,themselves, can be identified according to the techniques set forth inU.S. Pat. Appl. Pub. No. 2003/0190689A1, the entire contents of whichare hereby incorporated by reference.

In one aspect, the present invention provides for a method for treatingan EGFR-related disorder comprising predicting a response to anti-EGFRtherapy in a human subject in need of treatment by assaying a samplefrom the human subject prior to therapy with one or a plurality ofreagents that detect expression and/or activiation of predictivebiomarkers for an EGFR-related disorder such as cancer; determining apattern of expression and/opr activation of one or more of thepredictive biomarkers, wherein the pattern predicts the human subject'sresponse to the anti-EGFR therapy; and administering to a human subjectwho is predicted to respond positively to anti-EGFR treatment atherapeutically effective amount of a composition comprising an ABM ofthe present invention. As used herein, a human subject who is predictedto respondpositively to anti-EGFR treatment is one for whom anti-EGFRwill have a measurable effect on the EGFR-related disorder (e.g., tumorregression/shrinkage) and for whom the benefits of anti-EGFR therapy arenot outweighed by adverse effects (e.g., toxicity). As used herein, asample means any biological sample from an organism, particularly ahuman, comprising one or more cells, including single cells of anyorigin, tissue or biopsy samples which has been removed from organs suchas breast, lung, gastrointestinal tract, skin, cervix, ovary, prostate,kidney, brain, head and neck ,or any other other organ or tissue of thebody, and other body samples including, but not limited to, smears,sputum, secretions, cerebrospinal fluid, bile, blood, lymph fluid, urineand feces.

The composition comprising an ABM of the present invention will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disease or disorder being treated, the particular mammalbeing treated, the clinic condition of the individual patient, the causeof the disease or disorder, the site of delivery of the agent, themethod of administration, the scheduling of administration, and otherfactors known to medical practitioners. The therapeutically effectiveamount of the antagonist to be administered will be governed by suchconsiderations.

As a general proposition, the therapeutically effective amount of theantibody administered parenterally per dose will be in the range ofabout 0.1 to 20 mg/kg of patient body weight per day, with the typicalinitial range of antagonist used being in the range of about 2 to 10mg/kg. In one embodiment, the therapeutically effective amount is in therange of from about 1.0 mg/kg to about 15.0 mg/kg. In a more specificembodiment, the dose is in the range of from about 1.5 mg/kg to about 12mg/kg. In other embodiments, the dose is in the range of from about 1.5mg/kg to about 4.5 mg/kg, or from about 4.5 mg/kg to about 12 mg/kg. Thedose of the present invention may also be any dose within these ranges,including, but not limited to, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg,6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0 mg/kg, 11.5 mg/kg, 12.0mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0 mg/kg, 14.5 mg/kg, or15.0 mg/kg.

In a preferred embodiment, the ABM is an antibody, preferably ahumanized antibody. Suitable dosages for such an unconjugated antibodyare, for example, in the range from about 20 mg/m² to about 1000 mg/m².For example, one may administer to the patient one or more doses ofsubstantially less than 375 mg/m² of the antibody, e.g., where the doseis in the range from about 20 mg/m² to about 250 mg/m², for example fromabout 50 mg/m² to about 200 mg/m².

Moreover, one may administer one or more initial dose(s) of the antibodyfollowed by one or more subsequent dose(s), wherein the mg/m² dose ofthe antibody in the subsequent dose(s) exceeds the mg/m² dose of theantibody in the initial dose(s). For example, the initial dose may be inthe range from about 20 mg/m² to about 250 mg/m² (e.g., from about 50mg/m² to about 200 mg/m²) and the subsequent dose may be in the rangefrom about 250 mg/M² to about 1000 mg/M².

As noted above, however, these suggested amounts of ABM are subject to agreat deal of therapeutic discretion. The key factor in selecting anappropriate dose and scheduling is the result obtained, as indicatedabove. For example, relatively higher doses may be needed initially forthe treatment of ongoing and acute diseases. To obtain the mostefficacious results, depending on the disease or disorder, theantagonist is administered as close to the first sign, diagnosis,appearance, or occurrence of the disease or disorder as possible orduring remissions of the disease or disorder.

In the case of anti-EGFR antibodies used to treat tumors, optimumtherapeutic results have generally been achieved with a dose that issufficient to completely saturate the EGF receptors on the target cells.The dose necessary to achieve saturation will depend on the number ofEGF receptors expressed per tumor cell (which can vary significantlybetween different tumor types). Serum concentrations as low as 30 nMhave been effective in treating some tumors, while concentrations above100 nM may be necessary to achieve optimum therapeutic effect with othertumors. The dose necessary to achieve saturation for a given tumor canbe readily determined in vitro by radioimmunoassay orimmunoprecipiation.

In general, for combination therapy with radiation, one suitabletherapeutic regimen involves eight weekly infusions of an anti-EGFR ABMof the invention at a loading dose of 100-500 mg/M² followed bymaintenance doses at 100-250 mg/M² and radiation in the amount of 70.0Gy at a dose of 2.0 Gy daily. For combination therapy with chemotherapy,one suitable therapeutic regimen involves administering an anti-EGFR ABMof the invention as loading/maintenance doses weekly of 100/100 mg/M²,400/250 mg/M², or 500/250 mg/M² in combination with cisplatin at a doseof 100 mg/M² every three weeks. Alternatively, gemcitabine or irinotecancan be used in place of cisplatin.

The ABM of the present invention is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulinonary,and intranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antagonist may suitably beadministered by pulse infusion, e.g., with declining doses of theantagonist. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

One may administer other compounds, such as cytotoxic agents,chemotherapeutic agents, immunosuppressive agents and/or cytokines withthe antagonists herein. The combined administration includescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid. Also, the lipid carriermay be a detergent. As used herein, a detergent is any substance thatalters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR-1339 and TritonA-20).

Alternatively, the detergent may be an ionic detergent. An example of anionic detergent includes, but is not limited to, alkyltrimethylammoniumbromide.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a “liposome” is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

In yet another embodiment, the invention relates to an ABM according tothe present invention for use as a medicament, in particular for use inthe treatment or prophylaxis of cancer or for use in a precancerouscondition or lesion. The cancer may be, for example, lung cancer, nonsmall cell lung (NSCL) cancer, bronchioalviolar cell lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, chronic or acute leukemia, lymphocytic lymphomas, neoplasms ofthe central nervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme; astrocytomas, schwannomas, ependymomas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenomas, including refractory versions of any of the above cancers, ora combination of one or more of the above cancers. The precancerouscondition or lesion includes, for example, the group consisting of oralleukoplakia, actinic keratosis (solar keratosis), precancerous polyps ofthe colon or rectum, gastric epithelial dysplasia, adenomatousdysplasia, hereditary nonpolyposis colon cancer syndrome (HNPCC),Barrett's esophagus, bladder dysplasia, and precancerous cervicalconditions.

Preferably, said cancer is selected from the group consisting of breastcancer, bladder cancer, head & neck cancer, skin cancer, pancreaticcancer, lung cancer, ovarian cancer, colon cancer, prostate cancer,kidney cancer, and brain cancer.

Yet another embodiment is the use of the ABM according to the presentinvention for the manufacture of a medicament for the treatment orprophylaxis of cancer. Cancer is as defined above.

Preferably, said cancer is selected from the group consisting of breastcancer, bladder cancer, head & neck cancer, skin cancer, pancreaticcancer, lung cancer, ovarian cancer, colon cancer, prostate cancer,kidney cancer, and brain cancer.

Also preferably, said antigen binding molecule is used in atherapeutically effective amount from about 1.0 mg/kg to about 15 mg/kg.

Also more preferably, said antigen binding molecule is used in atherapeutically effective amount from about 1.5 mg/kg to about 12 mg/kg.

Also more preferably, said antigen binding molecule is used in atherapeutically effective amount from about 1.5 mg/kg to about 4.5mg/kg.

Also more preferably, said antigen binding molecule is used in atherapeutically effective amount from about 4.5 mg/kg to about 12 mg/kg.

Most preferably, said antigen binding molecule is used in atherapeutically effective amount of about 1.5 mg/kg.

Also most preferably, said antigen binding molecule is used in atherapeutically effective amount of about 4.5 mg/kg.

Also most preferably, said antigen binding molecule is used in atherapeutically effective amount of about 12 mg/kg.

In another embodiment, the present invention is directed to method oftreating an EGFR-related disorder in a mammal in need of treatmentthereof comprising administering to the mammal an ABM of of the presentinvention, wherein the treatment results in serum concentrations of saidABM between about 1 and about 500 μg/ml, for a period of at least 4weeks, and wherein the treatment does not cause a clinically significantlevel of toxicity in said mammal. In other embodiments, the serumconcentration is an amount selected from the group consisting of above 1μg/ml, above 25 μg/ml, above 50 μg/ml, above 100 μg/ml, above 200 μg/ml,above 300 μg/ml, above 400 μg/ml, above 500 μg/ml, between about 1 andabout 100 μg/ml, between about 1 and about 200 μg/ml, between about 1and about 300 μg/ml, between about 1 and about 400 μg/ml, and betweenabout 1 and about 500 μg/ml. In a preferred embodiment, the mammal is ahuman. In one embodiment the ABM is an antibody. In a preferredembodiment, the antibody is glycoengineered and has increasedFcgammaRIII binding compared to a non-glycoengineered form of theantibody.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an anti-EGFR antibody. The label orpackage insert indicates that the composition is used for treating thecondition of choice, such as a non-malignant disease or disorder, wherethe disease or disorder involves abnormal activation or production of anEGFR receptor and/or a EGFR-ligand, for example a benignhyperproliferative disease or disorder. Moreover, the article ofmanufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises a first antibodywhich binds EGFR and inhibits growth of cells which overexpress EGFR;and (b) a second container with a composition contained therein, whereinthe composition comprises a second antibody which binds EGFR and blocksligand activation of an EGFR receptor. The article of manufacture inthis embodiment of the invention may further comprises a package insertindicating that the first and second antibody compositions can be usedto treat a non-malignant disease or disorder from the list of suchdiseases or disorders in the definition section above. Moreover, thepackage insert may instruct the user of the composition (comprising anantibody which binds EGFR and blocks ligand activation of an EGFRreceptor) to combine therapy with the antibody and any of the adjuncttherapies described in the preceding section (e.g. a chemotherapeuticagent, an EGFR-targeted drug, an anti-angiogenic agent, animmunosuppressive agent, tyrosine kinase inhibitor, an anti-hormonalcompound, a cardioprotectant and/or a cytokine). Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

All patents, applications, and publications cited in this applicationare hereby incorporated by reference in their entirety.

EXAMPLES

Unless otherwise specified, references to the numbering of specificamino acid residue positions in the following Examples are according tothe Kabat numbering system. Unless otherwise noted, the materials andmethods used to make the antibodies described in these working examplesare in accordance with those set forth in the examples of U.S. patentapplication Ser. No. 10/981,738, which is herein incorporated byreference in its entirety.

Example 1

Materials and Methods

High Homology Acceptor Approach

The high homology antibody acceptor framework search was performed byaligning the rat ICR62 protein sequence to a collection of humangerm-line sequences and choosing that human sequence that showed thehighest sequence identity, while conserving all canonical residues on afunctional level. Here, the sequence 1-e from the VH1 family within theVBase database was chosen as the heavy chain framework acceptorsequence, and the A30 sequence from the VK1 family of the VBase databasewas chosen to be the framework acceptor for the light chain. On thesetwo acceptor frameworks the three complementarity determining regions(CDRs) and/or specificity-determining residues of those CDRs of the ratICR62 heavy and light variable domains were grafted. Since the framework4 region is not part of the variable region of the germ line gene, thealignment for that position was performed individually. The JH6 regionwas chosen for the heavy chain, and the JK2 region was chosen for thelight chain. Molecular modelling of the designed immunoglobulin domainrevealed one position outside of the Kabat CDR1 potentially requiringthe murine amino acid residues instead of the human ones outside of theCDR. Reintroduction of murine amino acid residues into the humanframework would generate so-called “back mutations.” For example, thehuman acceptor amino acid residue at Kabat position 27 (Glycine in 1-e)was back mutated to a tyrosine residue. To show the importance of theresidues for antigen binding, humanized antibody variants were designedthat either included or omitted the back mutations. The humanizedantibody light chain did not require any back mutations. After designingthe protein sequences, DNA sequences encoding these proteins weresynthesized as detailed below.

Mixed Framework Approach

To avoid the need for introducing back mutations at critical positions(critical to retain good antigen binding affinity or antibody functions)of the human acceptor framework, it was investigated whether frameworkregion 1 (FR1), framework regions 1 (FR1) and 2 (FR2) together, orframework region 3 (FR3) of a functionally humanized antibody could bereplaced by human antibody sequences already having donor residues, oramino acid residues that are fuictionally equivalent to donor residues,at important residue positions in the natural human germline sequence.For this purpose, the VH frameworks 1, 2 and 3 of the rat ICR62 VHsequence were aligned individually to human germ-line sequences. Here,highest sequence identity was not used for choosing acceptor frameworks;instead matching of several critical residues was performed. Thosecritical residues comprise the so-called canonical residues, and alsothose residues at position 27, 28, and 30 (Kabat numbering), which lieoutside of the CDR1 definition by Kabat, but often are involved inantigen binding. In addition, critical residues are those that showimportant interaction with the CDRs, as can be determined usingmolecular modelling. The IMGT sequence IGHV1-58 (Accession No. M29809),IGHV5-51 (Accession No. M99686), as well as the VBase sequence 1-02 fromthe VH1 family were chosen as suitable ones for replacing either FR1, 2,or 3. In brief: IGHV1-58 showed a promising pattern in the Kabatpositions 27 to 30, but does not fulfill the criteria for the canonicalposition 71. The IGHV5-51 has a matching residue 71, so its FR3 could beused. Also its FR1 is close to the desired FR1 sequence.

The 1-e of VH1 fulfilled all criteria apart from position 27. Sequence1-02 was considered acceptable for the FR1 and FR2 regions, but wouldrequire a back mutation in FR3.

After designing the protein sequences, DNA sequences encoding theseproteins were synthesized as detailed below. Using this approach backmutations were not necessary in most of the constructs of the heavychain, in order to retain good levels of antigen binding. The chronologyand the reasoning of the mixed framework constructs is explained in theresults section.

Synthesis of the Antibody Genes

After having designed the amino acid sequence of the humanized antibodyV region, the DNA sequence was generated. The DNA sequence data of theindividual frame work regions was found in the databases (e.g. IMGT orVBase) for human germ line sequences. The DNA sequence information ofthe CDR regions was taken from the published sequence of the rat ICR62antibody (see, e.g., PCT Publication WO 95/20045). With these sequences,the whole DNA sequence was virtually assembled. Having this DNA sequencedata, diagnostic restriction sites were introduced in the virtualsequence by introducing silent mutations, creating recognition sites forrestriction endonucleases. To obtain the physical DNA chain, genesynthesis was performed (see, e.g., Wheeler et al. 1995). In thismethod, oligonucleotides are designed from the genes of interest, such,that a series of oligonucleotides is derived from the coding strand, andone other series is from the non-coding strand. The 3′ and 5′ ends ofeach oligonucleotide (except the very first and last in the row) alwaysshow complementary sequences to two primers derived from the oppositestrand. When putting these oligonucleotides into a reaction buffersuitable for any heat stable polymerase, and adding Mg²⁺, dNTPs and aDNA polymerase, each oligonucleotide is extended from its 3′ end. Thenewly formed 3′ end of one primer then anneals with the next primer ofthe opposite strand, and extending its sequence further under conditionssuitable for template dependant DNA chain elongation. The final productwas cloned into a conventional vector for propagation in E. coli.

Antibody Production

For construction of the chimeric (i.e., fully rat V region and human Cregion) and humanized anti-EGFR light and heavy chain chain expressionvectors, human heavy and light chain leader sequences (for secretion)were added upstream of the variable region DNA sequences. Downstream ofthe variable regions, the constant regions of IgG1 for the heavy chainwere added, and the kappa constant region for the light chain usingstandard molecular biology techniques. The resulting full antibody heavyand light chain DNA sequences were subcloned into mammalian expressionvectors (one for the light chain and one for the heavy chain) under thecontrol of the MPSV promoter and upstream of a synthetic polyA site,each vector carrying an EBV OriP sequence.

Antibodies were produced by co-transfecting HEK293-EBNA cells with themammalian antibody heavy and light chain expression vectors using acalcium phosphate-transfection approach. Exponentially growingHEK293-EBNA cells were transfected by the calcium phosphate method. Forthe production of unmodified antibody, the cells were transfected onlywith antibody heavy and light chain expression vectors in a 1:1 ratio.For the production of the glycoengineered antibody, the cells wereco-transfected with four plasmids, two for antibody expression, one fora fusion GnTIII polypeptide expression, and one for mannosidase IIexpression at a ratio of 4:4:1:1, respectively. Cells were grown asadherent monolayer cultures in T flasks using DMEM culture mediumsupplemented with 10% FCS, and were transfected when they were between50 and 80% confluent. For the transfection of a T75 flask, 8 millioncells were seeded 24 hours before transfection in 14 ml DMEM culturemedium supplemented with FCS (at 10% V/V final), 250 μg/ml neomycin, andcells were placed at 37° C. in an incubator with a 5% CO2 atmosphereovernight. For each T75 flask to be transfected, a solution of DNA,CaCl2 and water was prepared by mixing 47 μg total plasmid vector DNAdivided equally between the light and heavy chain expression vectors,235 μl of a 1M CaCl2 solution, and adding water to a final volume of 469μl. To this solution, 469 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mMNa2HPO4 solution at pH 7.05 were added, mixed immediately for 10 sec andleft to stand at room temperature for 20 sec. The suspension was dilutedwith 12 ml of DMEM supplemented with 2% FCS, and added to the T75 inplace of the existing medium. The cells were incubated at 37° C., 5% CO2for about 17 to 20 hours, then medium was replaced with 12 ml DMEM, 10%FCS. The conditioned culture medium was harvested 5 to 7 dayspost-transfection centrifuged for 5 min at 1200 rpm, followed by asecond centrifugation for 10 min at 4000 rpm and kept at 4° C.

The secreted antibodies were purified by Protein A affinitychromatography, followed by cation exchange chromatography and a finalsize exclusion chromatographic step on a Superdex 200 column (AmershamPharmacia) exchanging the buffer to phosphate buffer saline andcollecting the pure monomeric IgG1 antibodies. Antibody concentrationwas estimated using a spectrophotometer from the absorbance at 280 nm.The antibodies were formulated in a 25 mM potassium phosphate, 125 mMsodium chloride, 100 mM glycine solution of pH 6.7.

Glycoengineered variants of the humanized antibody were produced byco-transfection of the antibody expression vectors together with aGnT-III glycosyltransferase expression vector, or together with aGnT-III expression vector plus a Golgi mannosidase II expression vector.Glycoengineered antibodies were purified and formulated as describedabove for the non-glycoengineered antibodies. The oligosaccharidesattached to the Fc region of the antibodies were analysed byMALDI/TOF-MS as described below.

Oligosaccharide Analysis

Oligosaccharides were enzymatically released from the antibodies byPNGaseF digestion, with the antibodies being either immobilized on aPVDF membrane or in solution.

The resulting digest solution containing the released oligosaccharideseither prepared directly for MALDI/TOF-MS analysis or was furtherdigested with EndoH glycosidase prior to sample preparation forMALDI/TOF-MS analysis.

Oligosaccharide Release Method for PVDF Membrane-Immobilized Antibodies

The wells of a 96-well plate made with a PVDF (Immobilon P, Millipore,Bedford, Mass.) membrane were wetted with 100 μl methanol and the liquidwas drawn through the PVDF membrane using vacuum applied to theMultiscreen vacuum manifold (Millipore, Bedford, Mass.). The PVDFmembranes were washed three times with 300 μl of water. The wells werethen washed with 50 μl RCM buffer (8M Urea, 360 mM Tris, 3.2 mM EDTA, pH8.6). Between 30-40 μg antibody was loaded in a well containing 10 μlRCM buffer. The liquid in the well was drawn through the membrane byapplying vacuum, and the membrane was subsequently washed twice with 50μl RCM buffer. The reduction of disulfide bridges was performed byaddition of 50 μl of 0.1M dithiothreitol in RCM and incubation at 37° C.for 1 h.

Following reduction, a vacuum was applied to remove the dithiothreitolsolution from the well. The wells were washed three times with 300 μlwater before performing the carboxymethylation of the cysteine residuesby addition of 50 μl 0.1M iodoacetic acid in RCM buffer and incubationat room temperature in the dark for 30 min.

After carboxymethylation, the wells were drawn with vacuum andsubsequently washed three times with 300 μl water. The PVDF membrane wasthen blocked, to prevent adsorption of the endoglycosidase, byincubating 100 μl of a 1% aqueous solution of polyvinylpyrrolidone 360at room temperature for 1 hour. The blocking reagent was then removed bygentle vacuum followed by three washes with 300 μl water.

N-linked oligosaccharides were released by addition of 2.5 mUpeptide-N-glycosydase F (recombinat N-Glycanase, GLYKO, Novato, Calif.)and 0.1 mU Sialidase (GLYKO, Novato, Calif.), to remove any potentialcharged monosaccharide residues, in a final volume of 25 μl in 2OmMNaHCO₃, pH7.0). Digestion was.performed for 3 hours at 37° C.

Oligosaccharide Release Method for Antibodies in Solution

Between 40 and 50 μg of antibody were mixed with 2.5 mU of PNGaseF(Glyko, U.S.A.) in 2 mM Tris, pH7.0 in a final volume of 25 microliters,and the mix was incubated for 3 hours at 37° C.

Use of Endoglycosidase H Digestion of PNGaseF-Released Oligosaccharidesfor the Assignment of Hybrid Bisected Oligosaccharide Structures toMALDI/TOF-MS Neutral Oligosaccharide Peaks

The PNGaseF released oligosaccharides were subsequently digested withEndoglycosidase H (EC 3.2.1.96). For the EndoH digestion, 15 mU of EndoH(Roche, Switzerland) were added to the PNGaseF digest (antibody insolution method above) to give a final volume of 30 microliters, and themix was incubated for 3 hours at 37° C. EndoH cleaves between theN-acetylglucosamine residues of the chitobiose core of N-linkedoligosaccharides. The enzyme can only digest oligomannose and mosthybrid type glycans, whereas complex type oligosaccharides are nothydrolyzed.

Sample Preparation for MALDI/TOF-MS

The enzymatic digests containing the released oligosaccharides wereincubated for a further 3 h at room after the addition of acetic acid toa final concentration of 150 mM, and were subsequently passed through0.6 ml of cation exchange resin (AG50W-X8 resin, hydrogen form, 100-200mesh, BioRad, Switzerland) packed into a micro-bio-spin chromatographycolumn (BioRad, Switzerland) to remove cations and proteins. Onemicroliter of the resulting sample was applied to a stainless steeltarget plate, and mixed on the plate with 1 μl of sDHB matrix. sDHBmatrix was prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid plus0.1 mg of 5-methoxysalicylic acid in 1 ml of ethanol/10 mM aqueoussodium chloride 1:1 (v/v). The samples were air dried, 0.2 μl ethanolwas applied, and the samples were finally allowed to re-crystallizeunder air.

MALDI/TOF-MS

The MALDI-TOF mass spectrometer used to acquire the mass spectra was aVoyager Elite (Perspective Biosystems). The instrument was operated inthe linear configuration, with an acceleration of 20kV and 80 ns delay.External calibration using oligosaccharide standards was used for massassignment of the ions. The spectra from 200 laser shots were summed toobtain the final spectrum.

Antigen Binding Assay

The purified, monomeric humanized antibody variants were tested forbinding to human epidermal growth factor receptor (EGFR, also referredto in the literature as HER-1 or ErbB1) on the A431 human epidermal cellline, using a flow cytometry-based assay. 200,000 cells (e.g., fromhuman A431 cell line) in 180 μl FACS buffer (PBS containing 2% FCS and 5mM EDTA) were transferred to 5 ml polystyrene tubes and 20 μl 10 foldconcentrated anti-EGFR antibody (primary antibody) samples (1-5000 ng/mlfinal concentration) or PBS only were added. After gently mixing thesamples, the tubes were incubated at 4° C. for 30 min in the dark.Subsequently, samples were washed twice with FACS buffer and pelleted at300×g for 3min. Supernatant was aspirated off and cells were taken up in50 μl FACS buffer and 2 μl secondary antibody (anti-Fc-specificF(ab′)2-FITC fragments (Jackson Immuno Research Laboratories, USA)) wasadded and the tubes were incubated at 4° C. for 30 min. Samples werewashed twice with FACS buffer and taken up in 500 μl of FACS buffer foranalysis by Flow Cytometry. Binding was determined by plotting thegeometric mean fluorescence against the antibody concentrations.

Binding of Monomeric IgG1 Glycovariants to FcγRIIIA-Expressing CHO CellLine

CHO cells were transfected by electroporation (280 V, 950 μF, 0.4 cm)with an expression vector coding for theFcgammaRIIIA-Val158 α-chain andthe γ-chain. Transfectants were selected by addition of 6 μg/mlpuromycin and stable clones were analyzed by FACS using 10 μlFITC-conjugated-anti-FcgammaRIII 3G8 monoclonal antibody (BDBiosciences, Allschwil/Switzerland) for 106 cells. Binding of IgG1 toFcgammaRIIIA-Val158-expressing CHO cells was performed. Briefly, theanti-FcgammaRIIIA 3G8 F(ab′)2 fragments (Ancell, Bayport, Minn. USA)were added at a concentration of 10 μg/ml to compete binding of antibodyglycovariants (3 μg/ml). The fluorescence intensity referring to thebound antibody variants was determined on a FACSCalibur (BD Biosciences,Allschwil /Switzerland).

ADCC assay

Human peripheral blood mononuclear cells (PBMC) were used as effectorcells and were prepared using Histopaque-1077 (Sigma Diagnostics Inc.,St. Louis, Mo. 63178 USA) following essentially the manufacturer'sinstructions. In brief, venous blood was taken with heparinized syringesfrom healthy volunteers. The blood was diluted 1:0.75-1.3 with PBS (notcontaining Ca⁺⁺ or Mg⁺⁺) and layered on Histopaque-1077. The gradientwas centrifuged at 400×g for 30 min at room temperature (RT) withoutbreaks. The interphase containing the PBMC was collected and washed withPBS (50 ml per cells from two gradients) and harvested by centrifugationat 300×g for 10 minutes at RT. After resuspension of the pellet withPBS, the PBMC were counted and washed a second time by centrifugation at200×g for 10 minutes at RT. The cells were then resuspended in theappropriate medium for the subsequent procedures.

The effector to target ratio used for the ADCC assays was 25:1 and 10:1for PBMC and NK cells, respectively. The effector cells were prepared inAIM-V medium at the appropriate concentration in order to add 50 μl perwell of round bottom 96 well plates. Target cells were human EGFRexpressing cells (e.g., A431, EBC-1, or LN229) grown in DMEM containing10% FCS. Target cells were washed in PBS, counted and resuspended inAIM-V at 0.3 million per ml in order to add 30,000 cells in 100 μl permicrowell. Antibodies were diluted in AIM-V, added in 50 μl to thepre-plated target cells and allowed to bind to the targets for 10minutes at RT. Then the effector cells were added and the plate wasincubated for 4 hours at 37° C. in a humidified atmosphere containing 5%CO2. Killing of target cells was assessed by measurement of lactatedehydrogenase (LDH) release from damaged cells using the CytotoxicityDetection kit (Roche Diagnostics, Rotkreuz, Switzerland). After the4-hour incubation the plates were centrifuged at 800×g. 100 μlsupernatant from each well was transferred to a new transparent flatbottom 96 well plate. 100 μl color substrate buffer from the kit wereadded per well. The Vmax values of the color reaction were determined inan ELISA reader at 490 nm for at least 10 min using SOFTmax PRO software(Molecular Devices, Sunnyvale, Calif. 94089, USA). Spontaneous LDHrelease was measured from wells containing only target and effectorcells but no antibodies. Maximal release was determined from wellscontaining only target cells and 1% Triton X-100. Percentage of specificantibody-mediated killing was calculated as follows:((x−SR)/(MR−SR)*100, where x is the mean of Vmax at a specific antibodyconcentration, SR is the mean of Vmax of the spontaneous release and MRis the mean of Vmax of the maximal release.

Example 2

Results and Discussion

Comparison of the binding to human EGF-receptor of antibody variantsI-HHA, I-HHB, I-HHC, I-HLA, I-HLB, I-HLC, I-HLA1, I-HLA2, I-HLA3,I-HLA4, I-HLA5, I-HLA6, I-HLA7, I-HLA8, I-HLA-9, I-HHD, I-HHE, I-HHF,and I-HHG, either complexed with the chimeric ICR62 light chain or withthe humanized ICR62 light chains (I-KA, I-KB, or I-KC) and the parental,chimeric antibody ch-ICR62 shows that all antibodies have within one logunit similar EC50 values. Only the I-HHA has strongly diminished bindingactivity (see FIG. 2). FIG. 1 shows the functional activity of theindividual chimeric ICR62 (ch-ICR62) polypeptide chains when combinedwith the humanized constructs I-HHC and I-KB, respectively. In thisexperiment, eiher the light chain, the heavy chain or both chainssimultaneously of the ch-ICR62 were replaced by the above mentionedhumanized constructs. This shows that the VH/VL interface formationseems to work as well in the rodent antibody as well as in the humanizedconstructs.

As shown in FIG. 2, the humanized heavy chain I-HHA could not restorebinding activity with either the I-KA, or the I-KB light chain. Sincethe I-HLA did show binding with both the I-KA, and the I-KB, the presentinventors concluded that the heavy chain of I-HHA is not functional inantigen binding. FIGS. 1 and 2, combined with FIG. 3, show that thelight chain constructs I-KA, I-KB, and I-KC show binding behaviorindistinguishable from the rodent counterpart. Variant I-KC does notpossess any back mutations, and additionally has its CDR1 partiallyhumanized, such that residues 24-29 can be derived from the humanacceptor sequence (A30 of VK1, as mentioned before).

In the series I-HHA, I-HHB, and I-HHC, only the latter two variantsshowed satisfactory binding behavior (FIGS. 2 and 3). Sequence analysisof the I-HHA revealed three potential amino acid residues responsiblefor this behavior: Lys33, His35, and Tyr50. Constructs that have Lys33replaced by tyrosine showed good binding, as well as constructs havingthe Tyr50 replaced by tryptophane. Only when these two residues werereplaced by alanine and glycine, respectively, was the binding lost.Since I-HHC did not show better binding than I-HHB, the presentinventors concluded that residues Asn60, Glu61, Lys64, and Ser65 neednot be of rodent origin; or they can be replaced by Ala, Gln, Gln, andGly, respectively. This procedure leads to a construct in which the CDR2is more humanized, since amino acid positions 60 to 65 are part of theKabat CDR definition, but there is no need to graft the rodent donorresidues for this antibody.

FIGS. 4 and 5 compare the constructs of the series I-HLA1, I-HLA2,I-HLA3, I-HLA4, I-HLA5, and I-HLA6. The best binding behavior wasobserved in the constructs ch-ICR62, I-HLA1, and IHLA2, with an EC50value of approx. 300 ng/ml. The other constructs had this valueincreased by a factor of two, and therefore have slightly reducedbinding activity. The first conclusion from this data is that, withinthe Kabat CDR1, the Lys33Tyr, and the Ile34Met substitutions weretolerated. These two positions are located within the Kabat definitionof CDR1, but outside of the Chothia CDR boundaries (that were based onstructural rather than sequence analysis). In the latter part of CDR1,then, at least some promiscuity is permitted.

The second conclusion is that, within CDR2, in addition to theabove-mentioned replacement of residues Asn60 and Glu61 by non-donorresidues, Asn60Ser, Glu61Pro, and Lys62Ser non-donor substitutionswithin the Kabat CDR were also allowed. These residues were derived fromthe human germ-line IGHV5-51 acceptor sequence, which was used as an FR3acceptor sequence. Constructs I-HLA3 and I-HLA4 differ from I-HLA1 andI-HLA2 only by the removal of the Phe27Tyr back mutation, and both theI-HLA3 and I-HLA4 constructs lose affinity compared to their parentalconstruct. Therefore, the third conclusion of the comparison of I-HLA1,I-HLA2, I-HLA3, I-HLA4, I-HLA5, and I-HLA6 is the involvement of Phe27in antigen binding, either directly or indirectly, via modifying theloop conformation of CDR1.

Variants I-HLA5 and I-HLA6 have the FR1 of I-HLA1 and I-HLA2,respectively, replaced by another germ-line acceptor sequence with thePhe27 naturally present (i.e., IGHV1-58). This could only be achieved bysimultaneously introducing several other mutations which are: Val2Met,Ala9Pro, and Ala16Thr. By doing so, the beneficial effect of(re-)introducing the Phe27 was again abrogated.

The I-HLA7 construct was assessed to determine whether the restorationof additional donor residues in the heavy chain CDR1 and CDR2 of theI-HLA6 construct would restore full binding activity as compared toICR62. As shown in FIG. 6, this was not the case.

As shown in FIGS. 7 and 8, two additional constructs, I-HLA8 and I-HLA9,were tested to determine if the full binding activity compared toch-ICR62 could be achieved. Starting from the I-HHB construct, the FR1regions were replaced by FR1 regions having maximal homology within theChothia CDR1 region. The I-HLA8 construct has the FR1 of the IGHV1-58sequence, and the I-HLA9 has the IGHV5-51 FR1 region. Both constructsbound the antigen at least as well as the ch-ICR62 antibody. The I-HLA8construct may, in fact, be even better, with the EC50 lowered by afactor of 2. The I-HLA8 construct has the same FR1 sequence as theI-HLA5 and I-HLA6 constructs and therefore has the same non-donorresidues (i.e., Val2Met, Ala9Pro, and Ala16Thr), suggesting that thepresence of these non-donor residues does not have a negative effect onbinding. Non-beneficial mutations occurring in the I-HLA5 and 6 arisefrom the combination of a VH1 FR1 paired with a VH5 FR3, which couldpotentially be compensated for by having a FR1 and a FR3 of the same VHfamily.

Shown in FIG. 8 are constructs that contain non-donor residues withinthe CDRs. Thus, these constructs are even further humanized within theCDRs because the non-donor residues occur in the human framework regionsthat were chosen for these constructs. The I-HHE (His35Ser), I-HHF(Thr58Asn) and I-HHG (Ser57Ala) constructs all have one residue withinthe CDR1 or CDR2 that is humanized (compared to the I-HHB construct).Construct I-HHD (Gly24Ala) was also assayed. I-HHF showed reducedbinding indicating the importance of Thr58. In contrast to the Kabat CDRresidue 58, amino acid 57 is more tolerant to substitutions, since theSer57Ala mutation apparently has no influence on binding (FIG. 8).

Since the FR3 region of IGHV5-51 seemed to show promising properties inthe I-HLA1 and 2 constructs, and the FR1 of the same germ-line sequenceproved to be useful in the I-HLA9 construct, the FR1, FR2, and FR3 ofIGHV5-51 was designed to be used together as an acceptor for loopgrafting.

Summary of the analysis of the canonical residues in humanized ICR62constructs:

-   -   VL: Kabat position 2: Ile probably required.        -   Kabat position 71: Ile or Phe allowed.    -   VH: Pos. 24, Gly, Thr, Ala, Val, Ser allowed.        -   Pos. 26, Gly allowed.        -   Pos. 29, Phe, Ile, Leu, Val, Ser allowed.        -   Pos. 34, Ile, Met, Leu, Val, Trp, Tyr, Thr allowed.        -   Pos. 71, Ala, Leu, Val, Thr allowed.        -   Pos 94, Arg, Gly, Asn, Lys, Ser, His, Thr, Ala allowed.            Results of the ADCC Experiments

FIG. 9 shows a comparison of the antibody mediated cellular cyotoxicity(ADCC) is shown for the various glycoforms of the chimeric ICR62antibody, as well as for the humanized variant I-HLA4. The differentglycoforms are marked by a label that either indicatesnot-glycoengineered (WT), Glycoform 1 (G1), or Glycoform 2 (G2). “G1”refers to glcyoengineering of the antibody by co-expression with GnTIII.“G2” refers to glycoengineering of the antibody by co-expression withGnTIII and ManII. “WT” refers to antibodies that were notglycoengineered. The light chain for all the humanized constructs is theI-KC variant, and was not labeled explicitly.

The chimeric, as well as the humanized antibody were improved in theirpotency and efficacy by the two different glycoengineering approaches.The ch-ICR62 construct performed slightly better than I-HLA4 for thewild-type or the glycoforms, respective. As seen in FIG. 4, whencomparing the affinities of the two antibodies towards their antigen,the ch-ICR62 had a twofold lower EC50 value. This difference in affinityis here reflected in differences in efficacy.

FIG. 10 shows a comparison of the antibody mediated cellular cyotoxicity(ADCC) for the non-glycoengineered (“wild-type”) and the G2 glycoform ofthe humanized ICR62 antibody constructs I-HHB and I-HLA7. The sameantibodies were applied to two different target cell lines. In panel Aof FIG. 10, the target cell line LN229 is used; and in panel B of FIG.10, the cell line A431 was used. The A431 cells are apparently moresusceptible towards antibody mediated cell killing than the LN229 cells.More importantly, the glycoengineering enhanced the potency of bothantibodies. This effect seemed to be more pronounced for the I-HLA7 thanfor the I-HHB. The percentage of cell killing at maximal antibodyconcentration for the I-HHB could be shifted from ˜30% to ˜40% byintroducing the G2 glycoengineered variant, when using the LN229 targetcell line. When using the A431 cell line, this value was apparentlyunchanged. This behavior was completely different for the I-HLA7antibody. Target cell killing at maximal antibody concentration wasshifted from about 10% to about 50%, for the LN229 cells, and from about40% to about 70% for the A431 cells by introducing the G2glycoengineering variants. In this case, despite having lower activityin the non glycoengineered antibody for the I-HLA7 relative to I-HHB,the ranking of activity is reversed for the glycoengineered antibodies.FIGS. 11 and 12 also show comparisons of non-glycoengineered forms (WT)and G2 glcyoforms of chimeric ICR62 and the humanized ICR62 antibodyconstructs I-HHB and I-HLA7.

Example 3 Preliminary Toxicity Study by Intravenous (Bolus)Administration to Cynomolgus Monkeys—Bioanalytical Analysis

Introduction

Glyco-Engineered Anti-EGFR Assay

This Bioanalytical Analysis describes the measurement of anti-EGFR insamples originating from cynomolgus monkeys following intravenous(bolus) administration of anti-EGFR (recombinant, glycoengineeredanti-EGFR antibody produced from transfected mammalian cells in culturewith antibody expression vectors harboring the heavy chain I-HHB and thelight chain I-KC genes as described above, and purified as describedabove) as described in the protocol set forth herein below. A total of78 monkey serum samples were stored frozen at about −20° C. until use.

The Bioanalytical methods used for the determination of anti-EGFR usedan ELISA method to measure serum concentrations of anti-EGFR. Acceptancecriteria were set at ±20% (±25% low QC) for precision and inaccuracy.

Materials and Methods

Obiective: The objective of this study was the assessment of systemictoxic potential of Glyco-mAb (anti-EGFR) intravenous (bolus)administration to Cynomolgus Monkeys followed by an 8-week recoveryperiod. TABLE 8 Animal model Cynomolgus Monkeys, accepted by regulatoryagencies, background data available. Justification for use of Theprimate was the non-rodent species of the primate choice because italone conserves two critical parameters: EGFR antigen recognition by thetest antibody, and test antibody Fc region recognition by immune systemFc receptors. Route Intravenous (Bolus), to simulate the conditions ofclinical administration.

TABLE 9 Treatment groups and dosages Group 1 2 3 CompoundGlyco-mAb(Anti-EGFR) Dosage (mg/kg/day) 1.5 4.5 12Rationale for Dosage Level Selection

1.5-7.5 mg/kg is the expected range for human studies (7.5 mg/kg beingthe corresponding dose for a similar compound in humans). TABLE 10Identity of treatment groups Number of Animal ID Dosage animals NumbersGroup Treatment (mg/kg/day) # Male Female Male Female 1 Glyco-mAb 1.5 11 623 590 (Anti-EGFR) 2 Glyco-mAb 4.5 1 1 461 462 (Anti-EGFR) 3Glyco-mAb 12 1 1 463 612 (Anti-EGFR)# Expressed in terms of the test substance as supplied.

TABLE 11 Animals Species Cynomolgus monkey (purpose bred). Age receivedApproximately 15 months. Weight range ordered 1.5 to 2.5 kg.

TABLE 12 Administration of Anti-EGFR Route Intravenous injection.Treated at Constant dosages in mg/kg/occasion. Volume dosage Calculatedin advance, based on the most recently recorded bodyweight. Individualdose 1 ml/kg/day volume Frequency Days 1, 8, 15 and 22, immediatelybefore feeding. Sequence By group. Dose sites Using left saphenousveins. Injection Bolus, new sterile disposable needle per animal.Formulation A record of the usage of formulation was maintained based onweights. This balance is compared with the expected usage as a check ofcorrect administration.

TABLE 13 Clinical observations Animals and their cage trays Visuallyinspected at least twice daily for evidence of reaction to treatment orill-health. Deviations from normal Nature and severity. recorded at thetime in Date and time of onset. respect of Duration and progress of theobserved condition. Physical examination Once each week for all animals.Daily records of cage trays For vomitus, blood, diarrhoea, etc.

TABLE 14 Dosing Frequency: Frequency 1. Immediately pre-dose. 2. ½ to 2hours after completion of dosing. 3. As late as possible in the workingday. Injection sites Daily.

TABLE 15 Toxicokinetics Day Animals Sample times hours after dosing. 1All animals 1, 4, 12, 24, 72, 120. 8 All animals Predose, 1 hourpost-dose (169 hours post Day 1 dose). 15 All animals Pre-dose, 1 hourpost-dose (337 hours post Day 1 dose). 22 All animals Pre-dose, 1 hourpost-dose (505 hours post Day 1 dose). 29 All animals 672 hours post Day1 dose.

TABLE 16 Samples Sample site Suitable vein. Anticoagulant/Sample Noanticoagulant/0.7 ml. volume Total number of samples 104. takenSeparation of serum By centrifugation at ambient temperature unlessotherwise indicated to provide a minimum of 0.3 ml, where possible.Storage of serum Appropriately labeled plastic tubes. Deep frozen(approximately −20° C.), while awaiting bioanalysis.

Histology TABLE 17 Tissue Fixation Standard 10% Neutral BufferedFormalin. Others Testes and epididymides: Initially in Bouin's fluid.Eyes: Initially in Davidson's fluid.

TABLE 18 Histology Processing All animals. Routine staining 4-5 μmsections stained with haematoxylin and eosin.Immunoassay Procedure

A plate was coated with 100 μl per well of coating solution (5 μl sheepanti-human IgG (monkey adsorbed IgG, the Binding Site, UK) added to11495 μl bicarbonate buffer (0.05M, pH9.6) and incubated forapproximately 2 hours at room temperature. The plate was washed 3 timeswith 400 μl per well of wash solution (PBS (Sigma Chemical Co., UK)0.01% (v/v) Triton-X100 (Sigma Chemical Co., UK)) and tapped dry.

Assay buffer (1% w/v BSA, Sigma Chemical Co., UK) was added at 200 μlper well and incubated for approximately 1 hour at room temperature. Theplate was washed 3 times with 400 μl per well of wash solution andtapped dry.

-   -   The calibration standards, Quality Controls (QC) and/or samples        were added at 100 μl per well and incubated for approximately 2        hours at room temperature, after which the plate was washed 3        times with 400 μl per well of wash solution and tapped dry.

The conjugate solution (6 μl goat anti-human IgG kappa-HRP conjugate(Bethyl Laboratories Inc., USA) added to 12 ml assay buffer) was addedat 100 μl per well and incubated for approximately 1 hour at roomtemperature. The plate was washed 3 times with 400 μl per well of washsolution and tapped dry.

Trimethylbenzidine (TMB; Europa Bioproducts, Ely, UK) was added at 100μl per well. The plate was covered and incubated for approximately 15minutes at room temperature. 100 μl of stop solution (0.5M HCI, Fisher,UK) was then added to each well. Absorbances were read at 450 nm(reference filter 630 nm) on a DYNATECH MRX microplate reader (MettlerInstruments, UK).

Results and Discussion

Test Sample Analysis

Concentrations of anti-EGFR were measured by an immunoassay method(ELISA) in 78 monkey serum samples generated according to the protocoldescribed herein above. These results are presented in Tables 19-21,below. TABLE 29 Serum concentration of anti-EGFR in monkey serum (μg/ml)following Intravenous administration of 1.5 mg/kg anti-EGFR on days 1,8, 15 and 22 (GROUP 1) Animal number Timepoint 1M 623 1F 590 Mean sd Day1 1 hour 33.42 30.86 32.14 1.8 Day 1 4 hours 27.33 27.49 27.41 0.1 Day 112 hours 13.09 17.01 15.05 2.8 Day 1 24 hours 9.656 9.468 9.562 0.1 Day1 72 hours 2.528 0.786 1.657 1.2 Day 1 120 hours 0.845 0.431 0.638 0.3Day 8 predose 0.538 0.287 0.413 0.2 Day 8 1 hour 30.02 19.07 24.55 7.7Day 15 predose 0.902 0.382 0.642 0.4 Day 15 1 hour 17.91 33.08 25.5010.7 Day 22 predose 1.065 0.595 0.830 0.3 Day 22 1hour 19.41 33.00 26.219.6 Day 1 672 hours 1.202 0.362 0.782 0.6sd standard deviation

TABLE 20 Serum concentration of anti-EGFR in monkey serum (μg/ml)following Intravenous administration of 5.0 mg/kg anti-EGFR on days 1,8, 15 and 22 (GROUP 2) Animal number Timepoint 2M 461 2F 462 Mean sd Day1 1 hour 32.45 29.51 30.98 2.1 Day 1 4 hours 32.39 29.57 30.98 2.0 Day 112 hours 28.05 25.88 26.97 1.5 Day 1 24 hours 23.70 23.78 23.74 0.1 Day1 72 hours 14.03 14.38 14.21 0.2 Day 1 120 hours 10.42 8.137 9.279 1.6Day 8 predose 4.672 3.683 4.178 0.7 Day 8 1 hour 25.91 31.06 28.49 3.6Day 15 predose 5.752 5.450 5.601 0.2 Day 15 1 hour 32.20 35.38 33.79 2.2Day 22 predose BLQ 6.497 3.249 — Day 22 1 hour 26.98 30.23 28.61 2.3 Day1 672 hours BLQ 4.845 2.423 —BLQ below limit of quantification (<0.195 μg/ml)sd standard deviationNote: BLQ entered as zero in calculations

TABLE 21 Serum concentration of anti-EGFR in monkey serum (μg/ml)following Intravenous administration of 15 mg/kg anti-EGFR on days 1, 8,15 and 22 (GROUP 3) Animal number Timepoint 3M 463 3F 612 Mean sd Day 11 hour 262.2 168.0 215.1 66.6 Day 1 4 hours 223.3 174.5 198.9 34.5 Day 112 hours 164.9 165.7 165.3 0.6 Day 1 24 hours 141.7 146.0 143.9 3.0 Day1 72 hours 99.54 86.64 93.09 9.1 Day 1 120 hours 86.64 69.08 77.86 12.4Day 8 predose 65.86 45.21 55.54 14.6 Day 8 1 hour 282.1 209.9 246.0 51.1Day 15 predose 98.43 71.21 84.82 19.2 Day 15 1 hour 385.9 231.4 308.7109.2 Day 22 predose 117.3 105.6 111.5 8.3 Day 22 1 hour 234.1 402.5318.3 119.1 Day 1 672 hours 127.5 122.9 125.2 3.3sd standard deviation

Example 4 Preliminary Toxicity Study by Intravenous (Bolus)Administration to Cynomolgus Monkeys—Toxicokinetics

Summary

Three groups of cynomolgus monkeys (1 male and 1 female per group) wereadministered intravenous bolus doses of anti-EGFR on Days 1, 8, 15 and22 of a 28-day toxicity study in order to assess the systemic exposureof the animals to anti-EGFR. Serum concentrations of anti-EGFR insamples collected up to 672 hours after the first dose were determinedby means of an immunoassay method. Pharmacokinetic analysis of serumconcentration-time data resulted in the following pharmacokineticparameters: TABLE 22 Dose C_(max) T_(max) AUC_(t) AUC CL V_(ss) kt_(1/2) (mg/kg) Animal (μg/mL) (h) (μg · h/mL) (μg · h/mL) (mL/h/kg)(mL/kg) (1/h) (h) 1.5 1M623 33.42 1 830.4  849.4 1.778 60.79 0.0214 32.51.5 1F590 30.86 1 748.4  774.9^(a) 1.962^(a) 57.85^(a) 0.0105^(a)66.0^(a) 4.5 2M461 32.45 1 2537  3005 1.488 133.6 0.0110 63.1 4.5 2F46229.57 4 2378  2719 1.663 133.2 0.0121 57.4 12 3M463 262.2 1 1831029870^(a) 0.4058^(a) 71.33^(a) 0.0056^(a) 124.3^(a) 12 3F612 174.5 415980 21400^(a) 0.5552^(a) 66.94^(a) 0.0082^(a) 84.4^(a)^(a)Value is an estimate as the data did not meet all the acceptancecriteria defined in Data Processing and should be treated with caution

The relationships between areas under the serum anti-EGFRconcentration-time curves (AUC₁₆₈) and dose level on Day 1 are presentedbelow: TABLE 23 Dose level Dose level AUC₁₆₈ ratio (mg/kg/occasion)ratio Males Females 1.5 1 1 1 4.5 3.0 3.1 3.2 12 8.0 22.0 21.4

The rate and extent of systemic exposure of monkeys to anti-EGFR,characterised by AUC₁₆₈, increased approximately proportionately withincreasing dose over the dose range 1.5 to 4.5 mg/kg/occasion but bymore than the proportionate dose increase over the dose range 4.5 to 12mg/kg/occasion on Day 1. At the highest dose (12 mg/kg/occasion) theAUC₁₆₈ was ca 2.8-fold higher than that predicted by a linearrelationship.

The extent (AUC₁₆₈) of systemic exposure of female monkeys to anti-EGFRwas generally similar to the exposure in male monkeys.

After repeated intravenous doses, the pre-dose serum concentrations ofanti-EGFR were generally higher than those values after a single doseand indicated accumulation of anti-EGFR in serum throughout the periodof the study.

The terminal half-life could not be estimated adequately for allanimals, but where it could be estimated was in the range 32.5 to 63.1hours, and appeared to increase with dose in male animals. Total serumclearance of anti-EGFR appeared to be independent of dose over the range1.5-4.5 mg/kg/occasion but was reduced at the top dose level in male andfemale monkeys.

In conclusion, the extent of systemic exposure of cynomolgus monkeys toanti-EGFR appeared to be characterised by non-linear (dose-dependent)kinetics over the dose range 1.5 to 12 mg/kg/occasion on Day 1 of theintravenous toxicity study. Increasing the dose of anti-EGFR above 4.5mg/kg/occasion is likely to result in a higher systemic exposure thanwould be predicted from a linear relationship, which is consistent withthe possibility of a capacity limited process for the elimination ofanti-EGFR.

In addition, the study also provided evidence that in general there wereno differences in the systemic exposure of male and female monkeys toanti-EGFR and that there was accumulation after repeated intravenousadministration.

Introduction

Three groups of one male and one female cynomolgus monkey wereadministered anti-EGFR by intravenous bolus injection, at dose levels of1.5, 4.5 and 12 mg/kg/occasion on Days 1, 8, 15 and 22 of a preliminarytoxicity study. Blood samples were taken from each animal at thefollowing time-points following administration on Day 1: 1, 4, 12, 24,72 and 120 hours post-dose. In addition, samples were taken pre-dose andat 1 hour post-dose on Days 8, 15 and 22 and at 672 hours after thefirst dose on Day 1. The separated serum was frozen at ca −20° C. priorto analysis of serum concentrations of anti-EGFR by an immunoassaymethod. Abbreviations AUC Area under the serum concentration-time curveto infinite time AUC₁₆₈ Area under the serum concentration-time curveduring a 168-hour dosing interval BLQ Below the limit of quantificationca Approximately CL Total serum clearance Cmax Maximum serumconcentration F Female k Terminal rate constant M Male t_(1/2) Terminalhalf-life Tmax Time at which Cmax occurred Vss Volume of distribution atsteady-stateAntibody Used for Study

Glyco-mAb (Anti-EGFR), an anti-EGFR antibody Fc-engineered for increasedFc-FcgammaRIII receptor binding affinity and increased ADCC, wasproduced, purified and characterized as described above. Briefly,antibody was produced by co-transfection of HEK-293-EBNA cells withplasmid DNA vectors for expression of I-HHB antibody heavy chain, I-KCantibody light chain, GnT-III and ManII. A linearly scaled-up version ofthe transfection method described above was employed, transfecting cellmonolayers cultured in roller bottles instead of T-flasks. An additionalflow-through anion-exchange chromatographic step using Q-sepharosematrix was included in the purification process immediately before thesize exclusion chromatographic step described above.

The glycosylation pattern of the Fc-engineered antibody was analyzed asdescribed above using MALDI/TOF-MS spectrometry of enzymaticallyreleased Fc-derived oligosaccharides. The oligosaccharide profile isshown in FIG. 23.

Binding to human EGFR and monkey EGFR was demonstrated by whole-cellbinding as described above using A431 and COS-7 cells, respectively, andFACS-based analysis. Binding curves are shown in FIGS. 24 and 25respectively.

Increased FcgammaRIII receptor binding resulting from the applied Fcengineering was demonstrated as described above using whole cell bindingto CHO cells engineered for surface expression of human FcgammaRIII andFACS-based analysis. Results are shown in FIG. 26. Additionally, theengineered antibody has equivalent degree of Fc-engineering to the“Glyco-2” antibody (75% on Fc-oligosaccharides being of non-fucosylatedtype) described elsewhere (Ferrara, C. et al., J Biol Chem. 2005 Dec. 5;[E-publication ahead of print]). Such Fc-engineered antibodies have upto 50-fold increased binding affinity for human FcgammaRIII relative toa standard non-Fc engineered antibody (Equilibrium dissociationconstanst of 15 and 150 nM for the 158V and 158F polymorphic variants ofthe human receptor vs. 750 and 5000 nM for the same receptor variants,respectively, when binding to non-Fc engineered human IgG1 antibodies).

ADCC was measured as described aboved using two target cell lines: A549human lung carcinoma cells and CYNOM-K1 cynomolgus monkey keratinocytecells. Results are shown in FIG. 27 and 28, respectively.

Data Processing

Pharmacokinetic parameters were calculated using the computer programWinNonlin Pro version 3.3 (Pharsight Corporation, USA).

All serum concentrations supplied as part of this study were reported to4 significant figures or 3 decimal places. Pharmacokinetic parameterswere reported as follows: Cmax , AUC₁₆₈, CL and Vss to 4 significantfigures; k to 4 decimal places; t_(1/2) to 1 decimal place.

Values that were BLQ (<0.195 μg/mL) were entered as zero in thepharmacokinetic processing.

Toxicokinetics

Maximum serum concentrations of anti-EGFR (Cmax) and their times ofoccurrence (Tmax) were the observed values. Areas under the serumanti-EGFR concentration-time curves within a 168-hour dosing interval(AUC₁₆₈), were estimated by the linear trapezoidal rule. In thecalculation of AUC₁₆₈ values the serum anti-EGFR concentrations at zerohours were estimated by back extrapolation using log-linear regressionanalysis, based on the first two sampling times, however, if the serumconcentration did not decline during this period then the serumconcentration at zero hours was considered to be equivalent to theconcentration at the first sampling time. Areas under the serumanti-EGFR concentration-time curves to infinite time (AUC), wereestimated by the following expression:AUC=AUC ₁₆₈ +Clast/kWhere Clast is the predicted serum concentration at the lastquantifiable sample point and k is the terminal rate constant.

Terminal rate constants (k) were estimated by fitting a linearregression of log concentration against time. For the estimate of k tobe accepted as reliable, the following criteria were imposed:

1. The terminal data points were apparently randomly distributed about asingle straight line (on visual inspection)

2. A minimum of 3 data points was available for the regression

3. The regression coefficient was ≧0.95, and the fraction of thevariance accounted for was ≧0.90

4. The interval including the data points chosen for the regression wasat least two-fold greater than the half-life itself

Terminal half-lives (t_(1/2)) were calculated as ln2/k. Total serumclearance (CL) was calculated as Dose/AUC. Volume of distribution atsteady-state (Vss) was calculated as Dose.AUMC/AUC2. Accumulation (R)was assessed as the ratio of the trough concentration following the lastdose (Day 22) to the trough concentration following the first dose(Day 1) i.e. serum concentration at 672 hours/serum concentration at 168hours (pre-dose on Day 8).

Results and Discussion

Blood samples were taken up to 120 hours after dosing on Day 1; atpre-dose and 1 hour post-dose on Days 8, 15 and 22, and at 672 hourspost dosing on Day 1 during a toxicity study to assess the systemicexposure of male and female monkeys to anti-EGFR following intravenousbolus administration of anti-EGFR at dose levels of 1.5, 4.5 and 12mg/kg/occasion on Days 1, 8, 15 and 22 of the study. Serumconcentrations of anti-EGFR in samples taken up to 168 hours post-doseare presented in Tables 27-29, and the mean serum concentration-timeprofiles are illustrated in FIGS. 18 and 19.

Pharmacokinetic parameters of anti-EGFR are presented in Table 50, andthe AUC₁₆₈ values are summarised below: TABLE 24 AUC168 Dose level (μg ·h/mL) (mg/kg/occasion) Males Females 1.5 830.4 748.4 4.5 2537 2378 1218310 15980

The times at which the maximum serum concentrations occurred (T_(max))were generally 1 hour post-dose (the first sample point) but occurred at4 hours post-dose (the second sample point) in females 2F462 (4.5 mg/kg)and 3F612 (12 mg/kg). However, for both these females, theconcentrations at 4 hours post-dose were very similar to thoseconcentrations at 1 hour post-dose and were probably within thevariability of the assay. Therefore the apparent delay in Tmax isunlikely to be of any significance.

Serum concentrations of anti-EGFR prior to the succeeding dose werequantifiable in all animals except male 2M461 on Day 22 (4.5mg/kg/occasion dose level) therefore, in general, animals werecontinuously exposed to quantifiable concentrations of anti-EGFR duringa dosing interval.

The relationships between areas under the serum anti-EGFRconcentration-time curves (AUC₆₈) and dose level on Day 1 are presentedbelow: TABLE 25 Dose level Dose level AUC₁₆₈ ratio (mg/kg/occasion)ratio Males Females 1.5 1 1 1 4.5 3.0 3.1 3.2 12 8.0 22.0 21.4

The rate and extent of systemic exposure of monkeys to anti-EGFR,characterised by AUC₁₆₈, increased approximately proportionately withincreasing dose over the dose range 1.5 to 4.5 mg/kg/occasion but bymore than the proportionate dose increase over the dose range 4.5 to 12mg/kg/occasion on Day 1. At the highest dose (12 mg/kg/occasion) theAUC₁₆₈ was ca 2.8-fold higher than that predicted by a linearrelationship (FIG. 20).

The extent (AUC₁₆₈) of systemic exposure of female monkeys to anti-EGFRwas generally similar to the exposure in male monkeys.

After repeated intravenous doses, the pre-dose serum concentrations ofanti-EGFR were generally higher than those values after a single dose(FIGS. 21-22) and indicated accumulation of anti-EGFR in serumthroughout the period of the study. This accumulation was generallylower in females than in males, except at the highest dose level. Theratios of the trough (pre-dose) concentrations following the last doseon Day 22 (672 hours post Day 1 dose) to the trough concentrationfollowing the first dose on Day 1 are presented in the Table 26, below:TABLE 26 Dose level R (mg/kg/occasion) Males Females 1.5 2.23 1.26 4.5 *1.32 12 1.94 2.72* Could not be calculated as trough concentration was BLQ

The terminal rate constants (k), and corresponding terminal half-lives(t_(1/2)), of anti-EGFR on Day 1 are presented in Table 30. The terminalhalf-life could not be estimated adequately for all animals, but whereit could be estimated was in the range 32.5 to 63.1 hours, and appearedto increase with dose in male animals. Total serum clearance ofanti-EGFR appeared to be independent of dose over the range 1.5-4.5mg/kg/occasion but was reduced at the highest dose level in male andfemale monkeys.

In conclusion, the extent of systemic exposure of cynomolgus monkeys toanti-EGFR appeared to be characterised by non-linear (dose-dependent)kinetics over the dose range 1.5 to 12 mg/kg/occasion on Day 1 of theintravenous toxicity study. Increasing the dose of anti-EGFR above 4.5mg/kg/occasion is likely to result in a higher systemic exposure thanwould be predicted from a linear relationship, which is consistent withthe possibility of a capacity limited process for the elimination ofanti-EGFR.

In addition, the study also provided evidence that in general there wereno differences in the systemic exposure of male and female monkeys toanti-EGFR and that there was accumulation after repeated intravenousadministration. TABLE 27 Serum concentrations (μg/ml) of anti-EGFR inmonkey serum following intravenous administration of 1.5 mg/kg anti-EGFRon Days 1, 8, 15 and 22 Animal number Timepoint 1M 623 1F 590 Day 1 1hour 33.42 30.86 Day 1 4 hours 27.33 27.49 Day 1 12 hours 13.09 17.01Day 1 24 hours 9.656 9.468 Day 1 72 hours 2.528 0.786 Day 1 120 hours0.845 0.431 Day 8 pre-dose 0.538 0.287 Day 8 1 hour 30.02 19.07 Day 15pre-dose 0.902 0.382 Day 15 1 hour 17.91 33.08 Day 22 pre-dose 1.0650.595 Day 22 1 hour 19.41 33.00 Day 1 672 hours 1.202 0.362

TABLE 28 Serum concentrations (μg/ml) of anti-EGFR in cynomolgus monkeyserum following intravenous administration of 4.5 mg/kg anti-EGFR onDays 1, 8, 15 and 22 Animal number Timepoint 2M 461 2F 462 Day 1 1 hour32.45 29.51 Day 1 4 hours 32.39 29.57 Day 1 12 hours 28.05 25.88 Day 124 hours 23.70 23.78 Day 1 72 hours 14.03 14.38 Day 1 120 hours 10.428.137 Day 8 pre-dose 4.672 3.683 Day 8 1 hour 25.91 31.06 Day 15pre-dose 5.752 5.450 Day 15 1 hour 32.20 35.38 Day 22 pre-dose BLQ 6.497Day 22 1 hour 26.98 30.23 Day 1 672 hours BLQ 4.845

TABLE 29 Serum concentrations (μg/ml) of anti-EGFR in cynomolgus monkeyserum following intravenous administration of 12 mg/kg anti-EGFR on Days1, 8, 15 and 22 Animal number Timepoint 1M 623 1F 590 Day 1 1 hour 262.2168.0 Day 1 4 hours 223.3 174.5 Day 1 12 hours 164.9 165.7 Day 1 24hours 141.7 146.0 Day 1 72 hours 99.54 86.64 Day 1 120 hours 86.64 69.08Day 8 pre-dose 65.86 45.21 Day 8 1 hour 282.1 209.9 Day 15 pre-dose98.43 71.21 Day 15 1 hour 385.9 231.4 Day 22 pre-dose 117.3 105.6 Day 221 hour 234.1 402.5 Day 1 672 hours 127.5 122.9

TABLE 30 Pharmacokinetic parameters of anti-EGFR on Day 1 of weeklyintravenous administration of anti-EGFR to cynomolgus monkeys DoseC_(max) T_(max) AUC_(t) AUC CL V_(ss) k t_(1/2) (mg/kg) Animal (μg/mL)(h) (μg · h/mL) (μg · h/mL) (mL/h/kg) (mL/kg) (1/h) (h) 1.5 1M623 33.421 830.4  849.4 1.778 60.79 0.0214 32.5 1.5 1F590 30.86 1 748.4 774.9^(a) 1.962^(a) 57.85^(a) 0.0105^(a) 66.0^(a) 4.5 2M461 32.45 12537  3005 1.488 133.6 0.0110 63.1 4.5 2F462 29.57 4 2378  2719 1.663133.2 0.0121 57.4 12 3M463 262.2 1 18310 29870^(a) 0.4058^(a) 71.33^(a)0.0056^(a) 124.3^(a) 12 3F612 174.5 4 15980 21400^(a) 0.5552^(a)66.94^(a) 0.0082^(a) 84.4^(a)^(a)Value is an estimate as the data did not meet all the acceptancecriteria defined in Data Processing and should be treated with cautionBlood Chemistry and Haematology

Blood samples were taken from the femoral vein of cynomolgous monkeysthat had been administered an intravenous bolus injection of GlycoMABanti-EGFR on days 1, 8, 15, and 22. Samples were taken from the limb notused for dose administration, following overnight deprivation of food(not decedents). Samples were examined at pretreatment, three days afterthe second dose, and on termination for the following parameters, usinglithium heparin as anticoagulant:

Alkaline phosphatase

Alanine amino-transferase

Aspartate amino-transferase

Bilirubin—total

Urea

Creatinine

Glucose

Cholesterol—total

Triglycerides

Sodium

Potassium

Chloride

Calcium

Phosphorus

Total protein

Protein electrophoretogram

Albumin/globulin ratio

Average normal cynomolgus monkey blood chemistry analysis data arepresented in Table 31. TABLE 31 Cynomolgus monkeys (originMauritius)-Blood Chemistry Parameter sex n 1% 5% 50% 95% 99% mean s.d.ALP M 949 837 1339 2147 3201 3899 2175.5 565.44 F 881 946 1342 2144 31633740 2164.1 552.82 ALP-N M 511 481 579 881 1453 1771 928.0 260.88 F 499427 546 846 1362 1694 879.5 240.24 ALT M 1489 24 30 50 87 127 53.7 19.07F 1407 23 28 46 84 111 49.3 18.65 AST M 1487 26 30 41 63 101 43.6 17.57F 1404 25 29 39 61 89 41.7 13.59 gGT M 663 95 118 178 292 342 188.552.47 F 641 81 102 153 232 266 158.6 38.38 LAP M 207 18 26 40 79 21745.1 28.25 F 205 15 20 35 62 89 37.1 12.73 GLDH M 159 8 10 17 35 12620.1 16.46 F 159 6 8 15 27 35 16.3 5.97 Bilirubin M 1494 1 1 3 8 11 3.82.04 F 1413 1 2 4 8 11 4.1 2.07 LDH M 99 160 596 808 1166 2029 838.3218.75 F 82 477 529 711 945 1021 715.0 117.07 CPK M 331 68 83 179 7131867 287.4 464.03 F 335 57 77 184 925 2628 309.7 534.02 Indir Bili M 591 2 4 10 11 4.2 2.15 F 57 1 2 4 5 7 3.6 1.13 Direct M 59 0 0 0 2 3 0.20.65 Bilirubin F 57 0 0 0 1 3 0.2 0.52 Bile Acids M 386 0.9 2.5 6.4 15.323.4 7.38 4.574 F 380 1.4 3.0 7.0 12.9 17.8 7.41 3.474 Urea M 1457 3.013.66 5.50 8.81 10.53 5.775 1.5710 F 1379 2.77 3.42 5.33 8.56 9.90 5.5591.5432 Creatinine M 1458 55 59 71 87 94 71.6 8.67 F 1383 56 60 72 87 8572.7 8.36 Glucose M 1455 2.22 2.64 3.71 5.21 6.37 3.809 0.8135 F 13802.23 2.65 3.63 5.18 6.34 3.735 0.7990 Cholesterol M 1455 1.69 1.93 2.683.55 3.96 2.706 1.4909 F 1382 1.83 2.15 2.86 3.69 4.05 2.885 0.4813 CholHDL M 45 1.26 1.34 1.79 2.23 2.59 1.784 0.3128 F 45 1.09 1.31 1.82 2.432.55 1.844 0.3179 Chol VLDL M 45 0.00 0.00 0.00 0.03 0.11 0.004 0.0175 F45 0.00 0.00 0.00 0.12 0.19 0.012 0.0370 NEFA M 132 0.10 0.28 0.98 1.842.44 0.994 0.4700 F 132 0.14 0.22 1.06 1.90 2.18 1.070 0.4704Triglycerides M 1453 0.26 0.32 0.53 0.86 1.30 0.561 0.2051 F 1374 0.260.34 0.57 0.90 1.14 0.587 0.1778 Ph Lipid M 64 1.65 1.68 2.18 2.91 3.152.254 0.3769 F 49 1.77 1.83 2.44 2.91 2.98 2.405 0.3267 Uric Acid M 17 00 0 8 8 1.1 2.34 F 17 0 0 0 1 1 0.4 0.51 Na M 1461 141 142 147 152 155146.8 3.00 F 1382 140 142 147 153 157 147.3 3.34 K M 1460 3.2 3.4 4.05.0 5.6 4.08 0.511 F 1382 3.2 3.3 4.0 4.9 5.4 4.01 0.484 Cl M 1461 102104 108 112 115 107.7 2.71 F 1382 102 104 108 113 116 108.4 2.83 Ca M1462 2.31 2.39 2.56 2.76 2.87 2.568 0.1176 F 1382 2.32 2.39 2.57 2.772.89 2.572 0.1168 Phos M 1172 1.16 1.40 1.93 2.43 2.69 1.921 0.3126 F1098 1.17 1.37 1.84 2.35 2.60 1.844 0.2978 Chol LDL M 45 0.54 0.62 1.201.87 1.92 1.253 0.3694 F 45 0.69 0.78 1.19 1.83 1.88 1.233 0.2906Bicarbonate M 288 7 10 17 22 25 16.5 3.51 F 283 6 10 16 22 25 15.9 3.81Total Protein M 1455 71 74 80 87 90 80.2 4.06 F 1381 71 74 81 89 92 81.24.32 Albumin M 346 34 38 43 46 49 42.6 2.67 (Chemical) F 342 36 38 43 4749 42.7 2.60 Albumin M 1089 33 36 45 51 54 44.3 4.46 F 1019 34 37 45 5255 44.7 4.58 Globulin M 289 31 32 36 41 45 36.3 2.83 F 285 30 32 37 4348 37.5 3.59 A/G Ratio M 340 0.79 0.98 1.17 1.39 1.45 1.172 0.1215(Chemical) F 336 0.84 0.95 1.14 1.34 1.42 1.143 0.1274 A/G Ratio M 10680.68 0.80 1.26 1.65 1.82 1.252 0.2522 F 998 0.67 0.79 1.26 1.66 1.811.247 0.2647 A1 Globulin M 1105 2 2 3 4 4 2.8 0.62 F 1035 2 2 3 4 5 2.80.64 A2 Globulin M 1105 3 3 4 6 7 4.2 0.92 F 1035 3 3 4 6 7 4.2 1.01beta M 1105 12 13 16 22 24 16.6 2.63 Globulin F 1035 12 13 16 22 25 16.82.85 gamma M 1105 8 9 12 17 18 12.6 2.26 Globulin F 1035 8 9 13 17 2013.1 2.52 Aldolase M 96 9 14 21 38 57 22.0 7.48 F 97 10 13 19 48 84 21.911.91 Plasm CHE M 17 4159 4159 5745 9160 9160 5919.8 1181.68 F 17 33713371 5869 8367 8367 5689.2 1512.98 CRP M 57 0.000 0.000 0.002 0.0130.026 0.0032 0.00414 F 56 0.000 0.000 0.002 0.004 0.007 0.0017 0.00167T3 M 40 1.90 1.90 2.50 3.38 3.58 2.537 0.4011 F 40 1.71 1.96 2.59 3.064.02 2.631 0.3799 T4 M 40 35 42 59 86 92 59.7 11.84 F 40 38 40 56 81 10758.6 14.37

Samples for haematological, peripheral blood analysis were taken fromthe femoral vein of cynomolgous monkey that had been administered anintravenous bolus injection of GlycoMAB anti-EGFR on days 1, 8, 15, and22. Samples were taken from the limb not used for dose administration,following overnight deprivation of food (not decedents). Samples wereexamined at pretreatment, three days after the second dose, and ontermination for the following parameters:

-   -   1) Using EDTA as anticoagulant—        -   HaematocritHaemoglobin concentration        -   Erythrocyte count        -   Reticulocytes        -   Mean cell haemoglobin        -   Mean cell haemoglobin concentration        -   Mean cell volume        -   Total leucocyte count        -   Differential leucocyte count        -   Platelet count        -   Abnormalities of the blood morphology            -   Anisocytosis            -   Microcytosis            -   Macrocytosis            -   Hypochromasia            -   Hyperchromasia    -   2) Using citrate as anticoagulant—        -   Prothrombin time        -   Activated partial thromboplastin time

Average normal cynomolgus monkey hematology analysis data are presentedin Table 32. TABLE 32 Cynomolgus monkeys (origin Mauritius)-HematologyParameter sex n 1% 5% 50% 95% 99% mean s.d. HCT M 1495 0.385 0.401 0.4430.488 0.512 0.4435 0.02776 F 1426 0.376 0.399 0.442 0.489 0.508 0.44240.02947 Haemoglobin M 1495 11.4 12.1 13.3 14.5 15.1 13.31 0.769 F 142611.2 11.9 13.2 14.5 15.1 13.21 0.855 RBC M 1495 5.67 6.04 6.74 7.51 7.926.744 0.4792 F 1426 5.58 5.96 6.71 7.45 7.73 6.707 0.4894 Retic (1) % M20 0.1 0.1 0.4 1.8 1.8 0.48 0.438 F 20 0.1 0.1 0.3 0.9 0.9 0.42 0.268Retic (2) % M 1476 0.21 0.27 0.49 0.95 1.58 0.551 0.3804 F 1408 0.220.28 0.54 1.06 1.60 0.595 0.2934 MCH M 1495 17.0 17.8 19.8 21.6 22.519.80 1.432 F 1425 16.7 17.7 19.7 21.8 22.6 19.74 1.216 MCHC M 1495 27.228.2 30.1 31.9 32.7 30.06 1.801 F 1425 27.0 27.9 29.9 31.8 32.6 29.881.174 MCV M 1495 57.9 60.5 65.8 71.4 73.5 65.88 3.278 F 1425 58.3 60.466.0 71.7 74.5 66.07 3.353 RDW M 280 12.6 12.9 14.4 16.1 16.7 14.400.934 F 285 12.4 12.8 14.2 15.7 16.3 14.21 0.879 WBC M 1507 5.61 6.6210.52 18.59 30.24 11.372 4.7766 F 1432 5.39 6.58 10.62 19.55 28.7911.637 4.7307 Neutrophils M 1507 0.88 1.28 3.49 10.24 16.27 4.319 3.1741F 1432 1.06 1.62 4.45 12.27 17.41 5.392 3.4777 Lymphocytes M 1507 2.192.96 5.53 9.81 15.91 6.021 3.4066 F 1432 2.16 2.67 4.86 8.55 13.95 5.2652.9997 Eosinophils M 1507 0.00 0.01 0.17 0.81 1.49 0.254 0.3127 F 14320.00 0.01 0.14 0.73 1.55 0.232 0.3188 Basophils M 1507 0.01 0.02 0.040.10 0.25 0.053 0.0627 F 1432 0.01 0.02 0.04 0.10 0.21 0.051 0.0540Monocytes M 1507 0.17 0.25 0.51 1.03 1.45 0.562 0.2575 F 1432 0.16 0.230.49 1.04 1.56 0.547 0.2705 Large Unstained M 1507 0.04 0.06 0.14 0.320.60 0.163 0.1330 Cells F 1432 0.04 0.06 0.13 0.29 0.50 0.148 0.1147Platelets M 1495 158 238 359 497 575 362.1 81.69 F 1426 181 234 359 496560 360.5 80.04 PT M 1481 9.6 9.9 10.8 12.0 14.8 10.88 0.877 F 1406 9.710.0 10.8 12.1 14.1 10.93 0.847 Act PTT M 1483 23.1 24.4 29.1 37.9 50.130.06 5.267 F 1408 22.8 24.4 29.4 37.4 47.2 30.19 5.185 Fibrinogen M 2651.61 1.86 2.61 3.51 4.88 2.664 0.6178 F 252 1.58 1.84 2.43 3.27 3.892.487 0.4545

The Biochemistry Cumulative Individual Values for the monkeys arepresented in Tables 33a-h, below: TABLE 33a Animal Group/ Occn. ALP ALTAST Bili Urea Creat Gluc Chol Number Sex Code U/L U/L U/L μmol/L mmol/Lμmol/L mmol/L mmol/L 615 1M PT 740 36 39 3 5.35 69 3.65 2.34 D11 743 3533 3 5.43 73 3.66 2.00 TERM 597 29 31 2 5.16 76 3.74 2.30 465 2M PT 64744 33 3 5.73 70 4.69 2.50 PD 775 47 36 2 3.60 73 4.50 2.72 D11 655 74 465 4.34 74 3.35 2.64 TERM 768 48 38 3 4.42 73 3.67 2.54 639 2M PD 741 2926 2 4.41 87 3.31 3.22 D11 629 29 28 3 3.87 99 2.64 2.99 TERM 599 34 222 3.62 83 3.46 2.45 613 3M PT 1003 37 31 5 3.80 90 5.72 2.61 D11 793 3629 4 4.45 80 3.28 2.70 TERM 931 34 32 5 5.16 83 2.78 2.46 631 4M PD 59034 37 2 3.43 83 3.92 2.49 D11 508 38 36 3 3.33 82 3.38 2.30 TERM 578 2525 2 4.00 89 3.70 2.26

TABLE 33b Animal Group/ Occn. Trig Na K Cl Ca Phos Total Prot Alb NumberSex Code mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L g/L g/L 615 1M PT0.24 146 4.7 108 2.54 1.77 91 35 D11 0.39 145 3.9 106 2.55 1.85 94 41TERM 0.34 147 4.2 107 2.47 1.63 87 38 465 2M PT 0.74 147 3.7 108 2.711.32 81 37 PD 0.93 147 4.1 109 2.70 1.54 84 46 D11 0.66 151 3.9 111 2.701.98 83 39 TERM 0.51 146 3.6 105 2.67 1.85 80 40 639 2M PD 0.27 146 4.2107 2.68 1.85 84 44 D11 0.32 150 4.8 108 2.81 2.07 85 42 TERM 0.38 1464.5 107 2.70 2.03 74 39 613 3M PT 0.47 151 4.5 106 2.75 1.85 83 41 D110.44 147 4.2 106 2.69 1.76 80 45 TERM 0.69 147 4.3 106 2.63 1.62 76 40631 4M PD 0.45 149 4.2 107 2.67 2.00 87 46 D11 0.77 151 5.0 112 2.682.00 84 39 TERM 0.64 150 4.7 107 2.77 1.89 86 46

TABLE 33c Animal Group/ Occn. a1 a2 Beta Gamma Number Sex Code g/L g/Lg/L g/L A/G Ratio Alb % a1 % a2 % 615 1M PT 3 5 25 23 0.63 39.0 3.4 5.1D11 3 4 22 25 0.77 43.1 3.0 4.5 TERM 3 4 21 21 0.78 43.5 3.3 4.9 465 2MPT 4 5 22 13 0.84 45.7 4.5 5.9 PD 3 4 18 12 1.21 54.9 3.6 5.0 D11 4 5 2015 0.89 46.7 4.9 6.4 TERM 3 5 20 11 1.00 50.5 4.0 5.8 639 2M PD 3 5 1714 1.10 52.9 3.5 5.8 D11 3 6 18 16 0.98 49.1 4.1 6.6 TERM 3 4 17 11 1.1152.2 3.7 6.0 613 3M PT 4 4 20 14 0.98 49.5 5.0 4.7 D11 3 3 17 13 1.2955.8 3.7 3.7 TERM 3 3 15 14 1.11 53.0 4.6 3.9 631 4M PD 3 4 18 16 1.1253.3 3.6 4.3 D11 4 4 19 18 0.87 46.4 4.4 4.4 TERM 3 4 19 15 1.15 53.13.4 4.3

TABLE 33d Animal Group/ Occn. Number Sex Code Beta % Gamma % 615 1M PT27.3 25.3 D11 23.2 26.2 TERM 24.4 23.9 465 2M PT 27.4 16.6 PD 21.8 14.7D11 24.0 17.9 TERM 25.5 14.2 639 2M PD 20.7 17.0 D11 21.5 18.7 TERM 23.214.9 613 3M PT 24.5 16.3 D11 21.0 15.8 TERM 20.0 18.5 631 4M PD 20.718.0 D11 22.8 22.0 TERM 22.1 17.1

TABLE 33e Animal Group/ Occn. ALP ALT AST Bili Urea Creat Gluc CholNumber Sex Code U/L U/L U/L μmol/L mmol/L μmol/L mmol/L mmol/L 614 1F PT687 71 50 3 4.92 60 4.45 2.77 D11 576 63 44 2 5.22 63 4.32 2.73 TERM 51759 41 3 4.78 70 3.98 2.83 652 1F PT 598 43 25 3 7.26 69 3.26 2.42 D11540 40 28 3 6.94 78 3.28 2.46 TERM 511 40 27 4 6.78 80 3.46 2.38 624 2FPD 533 56 35 3 3.85 73 3.91 2.31 D11 410 40 34 13 4.18 78 2.72 2.56 TERM432 41 25 3 3.70 78 3.30 2.16 632 3F PT 559 35 34 5 5.44 80 3.08 2.47D11 510 37 31 5 4.36 85 3.89 2.61 TERM 428 37 34 5 5.32 88 3.10 2.63 6404F PD 343 23 32 4 4.09 65 3.46 1.24 D11 292 25 28 4 4.12 63 2.69 1.13TERM 266 22 27 2 4.55 69 3.69 1.00

TABLE 33f Total Animal Group/ Occn. Trig Na K Cl Ca Phos Prot Alb NumberSex Code mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L g/L g/L 614 1F PT0.37 148 4.6 109 2.69 2.03 80 39 D11 0.33 147 4.1 109 2.72 2.08 82 43TERM 0.54 148 3.6 108 2.59 1.84 80 41 652 1F PT 0.57 147 4.3 105 2.581.52 78 35 D11 0.43 149 4.7 108 2.74 1.80 88 43 TERM 0.59 149 4.4 1082.59 1.51 80 40 624 2F PD 0.60 145 4.1 108 2.54 1.50 77 40 D11 0.51 1484.0 111 2.58 1.42 77 39 TERM 0.43 146 3.9 108 2.56 1.46 76 44 632 3F PT0.31 151 4.5 109 2.48 1.72 76 34 D11 0.34 149 4.6 109 2.58 1.85 80 39TERM 0.49 150 4.5 111 2.55 1.47 78 38 640 4F PD 0.36 144 4.8 111 2.311.45 68 29 D11 0.31 145 4.3 112 2.24 1.53 63 25 TERM 0.27 144 4.7 1082.20 1.33 60 25

TABLE 33g Animal Group/ Occn. a2 Beta Gamma A/G Number Sex Code a1 g/Lg/L g/L g/L Ratio Alb % a1 % a2 % 614 1F PT 4 5 20 13 0.95 49.2 4.4 5.9D11 3 4 19 13 1.10 52.7 3.3 4.9 TERM 3 4 18 13 1.05 51.7 3.8 5.2 652 1FPT 4 5 19 15 0.81 45.1 4.6 5.9 D11 3 5 20 17 0.96 49.3 3.4 5.5 TERM 4 518 14 1.00 49.4 4.7 6.2 624 2F PD 3 4 16 14 1.08 52.2 4.1 5.4 D11 4 4 1714 1.03 50.1 4.9 5.5 TERM 3 4 15 10 1.38 58.5 3.4 5.3 632 3F PT 4 4 1915 0.81 44.7 5.1 5.0 D11 3 4 17 16 0.95 49.2 4.2 4.7 TERM 4 4 17 15 0.9549.2 4.6 4.9 640 4F PD 4 4 17 15 0.74 42.2 5.7 5.6 D11 4 3 17 13 0.6640.1 6.6 5.5 TERM 4 3 16 13 0.71 41.4 6.4 4.6

TABLE 33h Animal Group/ Occn. Number Sex Code Beta % Gamma % 614 1F PT24.8 15.7 D11 23.1 16.0 TERM 22.8 16.5 652 1F PT 24.7 19.7 D11 22.3 19.4TERM 22.1 17.6 624 2F PD 20.7 17.6 D11 21.6 18.0 TERM 19.9 12.9 632 3FPT 24.9 20.2 D11 21.5 20.4 TERM 21.8 19.5 640 4F PD 24.9 21.6 D11 26.920.9 TERM 26.5 21.1

The Haematology Cumulative Individual Values for the monkeys arepresented in Table 34a-l, below: TABLE 34a Animal Group/ Occn. Hb RBCMCH MCHC MCV Number Sex Code Hct L/L g/dL ×10¹²/L Retic % pg g/dL fL 6151M PT 0.389 12.4 5.94 0.38 20.9 31.9 65.5 D11 0.366 11.5 5.59 0.76 20.631.4 65.5 TERM 0.381 11.8 5.91 0.25 20.0 31.0 64.5 465 2M PT 0.439 13.26.76 0.56 19.5 30.1 65.0 PTR PD 0.460 13.7 7.22 0.50 19.0 29.9 63.8 D110.391 11.7 6.11 1.62 19.1 29.9 64.0 TERM 0.441 13.6 6.93 0.65 19.7 30.963.7 639 2M PD 0.419 12.7 6.23 0.51 20.4 30.3 67.2 D11 0.400 11.7 5.990.52 19.6 29.3 66.7 TERM 0.388 12.2 5.70 1.14 21.4 31.4 68.1 613 3M PT0.461 14.1 6.79 0.48 20.7 30.5 67.8 PTR D11 0.396 12.7 6.05 0.92 21.032.1 65.4 TERM 0.410 12.9 6.23 0.49 20.8 31.5 65.9

TABLE 34b Animal Group/ Occn. WBC N L E Basophil Monocyte LUC Plt NumberSex Code ×10⁹/L ×10⁹/L ×10⁹/L ×10⁹/L ×10−9/L ×10−9/L ×10⁹/L ×10⁹/L 6151M PT 7.57 3.06 3.68 0.06 0.03 0.53 0.20 236 D11 7.56 2.78 4.35 0.060.02 0.21 0.13 284 TERM 7.93 3.77 3.52 0.06 0.02 0.49 0.07 254 465 2M PT14.19 1.78 10.37 1.24 0.05 0.55 0.20 302 PTR 13.69 3.69 8.25 0.93 0.040.47 0.29 PD 13.36 1.55 9.95 1.01 0.04 0.58 0.25 325 D11 12.26 4.70 5.631.27 0.04 0.51 0.11 403 TERM 15.45 1.54 11.57 1.65 0.07 0.42 0.20 356639 2M PD 10.02 5.21 3.42 0.90 0.01 0.39 0.10 306 D11 8.26 4.06 2.471.17 0.01 0.45 0.10 371 TERM 8.70 2.55 4.20 1.04 0.02 0.80 0.09 253 6133M PT 20.21 12.99 6.45 0.02 0.04 0.50 0.21 438 PTR 16.85 8.87 6.90 0.080.06 0.67 0.26 D11 10.85 6.11 4.15 0.03 0.02 0.41 0.12 441 TERM 23.2617.70 4.27 0.05 0.04 1.11 0.08 434

TABLE 34c Animal Group/ Occn. PT APTT Number Sex Code sec sec 615 1M PT11.3 37.3 D11 10.3 32.7 TERM 11.3 33.3 465 2M PT 10.0 33.9 PTR PD 9.926.1 D11 10.0 31.6 TERM 10.2 29.4 639 2M PD 10.6 22.6 D11 10.5 26.3 TERM10.3 28.9 613 3M PT 10.3 35.5 PTR D11 11.4 26.7 TERM 10.3 30.8

TABLE 34d Animal Group/ Occn. Number Sex Code Anisocytosis MicrocytosisMacrocytosis Hypochromasia Hyperchromasia 615 1M PT − − − − + D11 − − −− − TERM − − − − + 465 2M PT − − − − − PTR PD − − − − − D11 − − − − −TERM − − − − − 639 2M PD − − − − − D11 − − − − − TERM − − − − − 613 3MPT − − − − − PTR D11 − − − − + TERM − − − − −

TABLE 34e Animal Group/ Occn. Hb RBC MCH MCHC MCV Number Sex Code HctL/L g/dL ×10¹²/L Retic % pg g/dL fL 631 4M PD 0.460 13.6 7.11 0.43 19.129.5 64.6 D11 0.395 11.9 6.36 0.45 18.7 30.2 62.1 TERM 0.449 13.7 6.970.30 19.6 30.5 64.4 614 1F PT CTD CTD CTD CTD CTD CTD CTD PTR D11 0.40413.1 6.33 0.57 20.6 32.3 63.8 TERM 0.424 13.0 6.59 0.68 19.7 30.7 64.3652 1F PT 0.390 11.3 5.72 1.15 19.8 29.1 68.2 D11 0.374 11.9 5.55 1.0621.4 31.7 67.4 TERM 0.384 11.5 5.71 0.58 20.2 30.0 67.2 624 2F PD 0.40711.8 7.14 0.73 16.6 29.0 57.1 D11 0.377 10.8 6.69 0.48 16.1 28.6 56.3TERM 0.401 11.7 6.99 1.07 16.7 29.1 57.4

TABLE 34f Animal Group/ Occn. WBC N L E LUC Plt Number Sex Code ×10⁹/L×10⁹/L ×10⁹/L ×10⁹/L Basophil Monocyte ×10⁹/L ×10⁹/L 631 4M PD 10.532.82 6.36 0.62 0.02 0.58 0.13 378 D11 7.97 2.86 3.81 0.61 0.01 0.53 0.16424 TERM 8.08 1.73 5.04 0.63 0.02 0.60 0.05 384 614 1F PT CTD CTD CTDCTD CTD CTD CTD CTD PTR 11.32 3.47 6.36 0.17 0.05 0.96 0.31 D11 9.704.74 3.73 0.09 0.02 0.84 0.28 429 TERM 9.64 3.65 5.09 0.13 0.01 0.620.13 414 652 1F PT 10.66 3.21 6.05 0.42 0.03 0.80 0.15 373 D11 12.015.53 5.20 0.34 0.02 0.78 0.13 380 TERM 11.88 7.59 3.24 0.35 0.02 0.610.08 338 624 2F PD 9.06 3.10 5.02 0.41 0.02 0.33 0.18 362 D11 7.82 5.142.06 0.14 0.02 0.34 0.13 353 TERM 11.69 4.33 5.46 0.96 0.02 0.76 0.16426

TABLE 34g Animal Group/ Occn. PT APTT Number Sex Code sec sec 631 4M PD10.9 29.3 D11 11.3 27.9 TERM 10.8 32.1 614 1F PT CTD CTD PTR D11 10.232.1 TERM 10.5 29.3 652 1F PT 10.2 33.0 D11 9.6 27.9 TERM 10.3 30.3 6242F PD 10.6 27.1 D11 10.6 29.7 TERM 10.8 33.3

TABLE 34h Animal Group/ Occn. Number Sex Code Anisocytosis MicrocytosisMacrocytosis Hypochromasia Hyperchromasia 631 4M PD − − − − − D11 − − −− − TERM − − − − − 614 1F PT CTD CTD CTD CTD CTD PTR D11 − − − − − TERM− − − − − 652 1F PT − − − − − D11 − − − − − TERM − − − − − 624 2F PD − +− − − D11 − + − − − TERM + + − − −

TABLE 34i Animal Group/ Occn. Hb RBC MCH MCHC MCV Number Sex Code HctL/L g/dL ×10¹²/L Retic % pg g/dL fL 632 3F PT 0.416 12.4 6.36 0.83 19.629.9 65.4 PTR 0.410 12.2 6.29 0.64 19.5 29.8 65.3 D11 0.392 12.2 6.190.73 19.7 31.0 63.4 TERM 0.412 12.3 6.46 0.55 19.1 29.9 63.8 640 4F PD0.398 11.8 5.81 0.95 20.3 29.7 68.5 D11 0.369 11.0 5.30 1.17 20.7 29.869.6 TERM 0.401 11.9 5.58 0.83 21.3 29.6 71.9

TABLE 34j Animal Group/ Occn. WBC N L E LUC Plt Number Sex Code ×10⁹/L×10⁹/L ×10⁹/L ×10⁹/L Basophil Monocyte ×10⁹/L ×10⁹/L 632 3F PT 18.1210.58 5.61 1.07 0.04 0.61 0.21 335 PTR 15.16 5.99 7.24 0.95 0.03 0.630.31 308 D11 10.23 4.69 4.15 0.66 0.01 0.48 0.23 348 TERM 13.04 6.894.62 0.44 0.03 0.93 0.13 321 640 4F PD 12.49 8.29 2.91 0.46 0.02 0.640.17 567 D11 13.71 10.31 2.18 0.34 0.01 0.74 0.12 578 TERM 11.49 5.414.18 0.63 0.03 1.12 0.12 555

TABLE 34k Animal Group/ Occn. PT APTT Number Sex Code sec sec 632 3F PT10.6 47.8 PTR 10.6 44.7 D11 10.2 33.1 TERM 10.6 37.2 640 4F PD 12.0 25.8D11 12.4 28.0 TERM 12.8 30.1

TABLE 34l Animal Group/ Occn. Number Sex Code Anisocytosis MicrocytosisMacrocytosis Hypochromasia Hyperchromasia 632 3F PT — — — — — PTR — — —— — D11 — — — — — TERM — — — — — 640 4F PD — — — — — D11 — — — — — TERM— — — — —Microscopic Pathology—Treatment-Related Findings

Pericholangitis (inflammation of connective tissue around the bile duct)was reported in the female monkey dosed at 12 mg/kg/day, but not in anyother female or male monkeys. This finding may be related to treatmentwith Glyco-mAb (Anti-EGFR), but with such small numbers of animals thesignificance is uncertain. All other findings were considered to beincidental and of no toxicological significance.

Macropathology and Histopathology

The summary of histopatholigical for all animals tested is set forth inTable 35, below: TABLE 35 Histopathology - group distribution andseverity of findings for all animals Group 1 2 3 Compound -GLYCO-MAB(ANTI-EGFR)- Dosage 1.5 4.5 12 Number of Animals Affected Sex MaleFemale Group 1 2 3 1 2 3 Number Organ/Tissue Examined 1 1 1 1 1 1 ColonNo. Examined 1 1 1 1 1 1 Heart No. Examined 1 1 1 1 1 1 Kidneys No.Examined 1 1 1 1 1 1 Cortical Lymphocytic Minimal 1 1 0 0 1 1Infiltration Slight 0 0 0 1 0 [?] Total 1 1 0 1 1 1 Left Cephalic No.Examined 0 0 0 0 0 0 Left Saphenous No. Examined 1 1 1 1 1 1  EpidermalHyperplasia Minimal 0 0 0 1 0 0 Total 0 0 0 1 0 0 Liver No. Examined 1 11 1 1 1  Inflammatory Cell Foci Minimal 1 1 1 1 1 1 Total 1 1 1 1 1 1 Bile Duct Proliferation Minimal 0 0 0 0 0 1 Total 0 0 0 0 0 1 Hepatocyte Vacuolation - Minimal 0 1 0 0 0 0 Median Cleft Total 0 1 0 00 0  Pericholangitis Minimal 0 0 0 0 0 1 Total 0 0 0 0 0 1 Lungs &Bronchi No. Examined 1 1 1 1 1 1  Bronchi/Bronchioles - Slight 1 0 0 0 00 Mucosal/Submucosal Inflammatory Cells Total 1 0 0 0 0 0  AlveolarMacrophages Minimal 0 1 0 0 0 1 Total 0 1 0 0 0 1  Perivascular Minimal0 0 1 0 0 1 Inflammatory/Lymphoid Cells Total 0 0 1 0 0 1  LymphoidAggregates Minimal 0 0 1 0 0 0 Total 0 0 1 0 0 0 Oesophagus No. Examined1 1 1 1 1 1  Lymphoid Aggregates Minimal 0 0 0 0 0 1 Total 0 0 0 0 0 1Ovaries No. Examined 0 0 0 1 1 1  Follicular Cyst(S) Present 0 0 0 1 0 0Total 0 0 0 1 0 0  Prominent Corpora Lutea Present 0 0 0 0 1 0 Total 0 00 0 1 0 Pancreas No. Examined 1 1 1 1 1 1  Acinar Atrophy Minimal 0 0 11 0 1 Total 0 0 1 1 0 1  Lymphoid Aggregates Minimal 0 0 0 1 0 0 Total 00 0 1 0 0 Right Cephalic No. Examined 0 0 0 0 0 0 Right Saphenous No.Examined 0 0 0 0 0 0 Skin (Protocol) No. Examined 1 1 1 1 1 1  EpidermalHyperplasia Minimal 0 0 0 0 0 1 Moderate 0 0 0 1 0 0 Total 0 0 0 1 0 1Spinal Cord No. Examined 1 1 1 1 1 1  Haemorrhage Minimal 0 0 1 1 1 1Slight 0 1 0 0 0 0 Total 0 1 1 1 1 1 Spleen No. Examined 1 1 1 1 1 1Sternum & Marrow No. Examined 0 0 0 0 0 0 Stomach No. Examined 1 1 1 1 11 Testes No. Examined 1 1 1 0 0 0  Immaturity Present 1 1 1 0 0 0 Total1 1 1 0 0 0 Thymus No. Examined 1 1 1 1 1 1  Cyst(S) Present 0 0 0 0 1 0Total 0 0 0 0 1 0  Involution/Atrophy Minimal 0 0 0 0 1 0 Total 0 0 0 01 0 Urinary Bladder No. Examined 1 1 1 1 1 1 Uterine Cervix No. Examined0 0 0 1 1 1  Epithelial Mucification Present 0 0 0 1 1 1 Total 0 0 0 1 11 Uterus No. Examined 0 0 0 1 1 1  Congestion Minimal 0 0 0 0 1 0 Total0 0 0 0 1 0 Caecum No. Examined 1 0 0 1 0 0  Prominent SubmucosalMinimal 1 0 0 1 0 0 Adipose Tissue Total 1 0 0 1 0 0 Fallopian Tube No.Examined 0 0 0 1 1 1 Ln Mesenteric No. Examined 0 0 0 1 0 0  IncreasedPigmented Slight 0 0 0 1 0 0 Macrophages Total 0 0 0 1 0 0Individual Findings for All Animals

The pathology observations for individual animals are set forth in Table36, below: TABLE 36 Macropathology and histopathology - individualfindings for all animals Group 1 2 3 Compound -GLYCO-MAB (ANTI-EGFR)-Dosage 1.5 4.5 12 Pathology Observations Sex Male Dose Group 1 AnimalNo. 0623 Study week of Sacrifice 11 Terminal body weight 2715.0 gramsStudy day of sacrifice 77 NECROPSY HISTOPATHOLOGY Caecum: Caecum:-Raised Area(S); Mucosal Aspect, -Prominent Submucosal Adipose Tissue, -Multiple, Up To 3 mm. Minimal, Focal Colon: Colon: -Raised Area(S);Mucosal Aspect, No Significant Lesion Multiple, Up To 2 mm. Kidneys:-Cortical Lymphocytic Infiltration, -Minimal Liver: Liver: -Median CleftPale Area(S); One, -Inflammatory Cell Foci, -Minimal Subcapsular, 3 mm.Lungs & Bronchi: -Bronchi/Bronchioles Mucosal/Submucosal InflammatoryCells, - Slight Stomach: Stomach: -Corpus Raised Area(S); Mucosa,One, >No Significant Lesion Near To Antrum, 3 mm. Testes: -Immaturity,-Present Sex Male Dose Group 2 Animal No. 0461 Study week of Sacrifice18 Terminal body weight 2573.0 grams Study day of sacrifice 125 NECROPSYHISTOPATHOLOGY Kidneys -Cortical Lymphocytic Infiltration, -MinimalLiver: Liver -Median Cleft Pale Area(S); One, -Inflammatory Cell Foci,-Minimal Subcapsular, 4 mm. -Hepatocyte Vacuolation - Median Cleft, -Minimal Lungs & Bronchi: Lungs & Bronchi: -Incomplete Collapse; RightLobes. -Alveolar Macrophages, -Minimal Spinal Cord: -Haemorrhage,-Slight, Multi-Focal Testes: -Immaturity, -Present Sex Male Dose Group 3Animal No. 0463 Study week of Sacrifice 18 Terminal body weight 2919.0grams Study day of sacrifice 125 NECROPSY HISTOPATHOLOGY Liver:-Inflammatory Cell Foci, -Minimal Lungs & Bronchi: -PerivascularInflammatory/Lymphoid Cells, - Minimal -Lymphoid Aggregates, -Minimal,Focal Pancreas: -Acinar Atrophy, -Minimal, Focal Spinal Cord:-Haemorrhage, -Minimal Testes: -Immaturity, -Present ***Animal has nogross observations recorded*** Sex FeMale Dose Group 1 Animal No. 0590Study week of Sacrifice 11 Terminal body weight 3176.0 grams Study dayof sacrifice 77 NECROPSY HISTOPATHOLOGY Caecum: Caecum: -Raised Area(S);Mucosal Aspect, -Prominent Submucosal Adipose Tissue, - Multiple, Up To2 mm. Minimal, Multi-Focal Colon: Colon: -Raised Area(S); MucosalAspect, >No Significant Lesion Multiple, Up To 2 mm. Kidneys: -CorticalLymphocytic Infiltration, -Slight, Focal Left Saphenous: -EpidermalHyperplasia, -Minimal Liver: Liver: -Median Cleft Pale Area(S); One,-Inflammatory Cell Foci, -Minimal Subcapsular, 3 mm. Ln Mesenteric: LnMesenteric: -Congested, Minimal -Increased Pigmented Macrophages,-Slight Ovaries: Ovaries: -Cyst(S); Left, One, Clear Fluid-Filled,-Follicular Cyst(S), -Present 4 mm. Pancreas: -Acinar Atrophy, -Minimal-Lymphoid Aggregates, -Minimal Skin (Protocol): -Epidermal Hyperplasia,-Moderate Spinal Cord: -Haemorrhage, -Minimal Spleen: Spleen: -CapsuleThickened; Area, Diffuse. >No Significant Lesion Uterine Cervix:-Epithelial Mucification, -Present Sex Female Dose Group 2 Animal No.0462 Study week of Sacrifice 18 Terminal body weight 2910.0 grams Studyday of sacrifice 125 NECROPSY HISTOPATHOLOGY Kidneys: -CorticalLymphocytic Infiltration, -Minimal, Focal Liver: -Inflammatory CellFoci, -Minimal Lungs & Bronchi: Lungs & Bronchi: -Incomplete Collapse;Left Lobes. No Significant Lesion Ovaries: Ovaries: -Raised Area(S); OneOn Each, Left, -Prominent Corpora Lutea, -Present 3 mm; Right, 2 mm.(Follicles) Spinal Cord: -Haemorrhage, -Minimal Thymus: Thymus: -Small;1.066 g. -Cyst(S), -Present -Involution/Atrophy, -Minimal UterineCervix: -Epithelial Mucification, -Present Uterus: Uterus: -Congested,Minimal -Congestion, -Minimal Sex Female Dose Group 3 Animal No. 0612Study week of Sacrifice 18 Terminal body weight 2934.0 grams Study dayof sacrifice 125 NECROPSY HISTOPATHOLOGY Kidneys: -Cortical LymphocyticInfiltration, -Minimal, Focal Liver: Liver: -Median Cleft Pale Area(S);One, -Inflammatory Cell Foci, -Minimal Subcapsular, 3 mm. -Bile DuctProliferation, -Minimal -Cyst(S); Within Cleft, One, Dark Fluid--Pericholangitis, -Minimal Filled, Green, 2 mm. Lungs & Bronchi: Lungs &Bronchi: -Incomplete Collapse; Left Lobes. -Alveolar Macrophages,-Minimal -Perivascular Inflammatory/Lymphoid Cells, - MinimalOesophagus: -Lymphoid Aggregates, -Minimal Pancreas: -Acinar Atrophy,-Minimal, Focal Skin (Protocol): -Epidermal Hyperplasia, -Minimal SpinalCord: -Haemorrhage, -Minimal Uterine Cervix: -Epithelial Mucification,-Present

Individual body weights of the cynomolgus monkeys are presented in Table37, below: TABLE 37 Bodyweights: Individual Values Animal Bodyweight(kg) on Day No. −17 −9 1* 8* 15* 22* 29 36 Group 1: GA201-ge, 1.5mg/kg/occasion 623m 2.67 2.65 2.69 2.72 2.76 2.62 2.72 2.65 2.71 (−0.02)(+0.04) (+0.03) (+0.04) (−0.14) (+0.10) (−0.07) (+0.06) 590f 2.98 2.922.97 3.14 3.13 3.01 3.06 3.00 3.19 (−0.06) (+0.05) (+0.17) (−0.01)(−0.12) (+0.05) (−0.06) (+0.19) Group 2: GA201-ge, 4.5 mg/kg/occasion461m 2.50 2.56 2.53 2.47 2.53 2.53 2.53 2.61 2.53 (+0.06) (−0.03)(−0.06) (+0.06) NC NC (+0.08) (−0.08) 462f 2.87 2.96 2.91 2.82 2.95 2.892.88 2.79 2.91 (+0.09) (−0.05) (−0.09) (+0.13) (−0.06) (−0.01) (−0.09)(+0.12) Group 3: GA201-ge, 12 mg/kg/occasion 463m 2.69 2.81 2.86 2.742.81 2.81 2.81 2.81 2.75 (+0.12) (+0.05) (−0.12) (+0.07) NC NC NC(−0.06) 612f 2.84 2.96 2.92 2.98 3.06 3.01 3.01 2.94 2.91 (+0.12)(−0.04) (+0.06) (+0.08) (−0.05) NC (−0.07) (−0.03) Animal Bodyweight(kg) on Day Weight change (kg) No. 50 57 64 71 77 D 1 to 71 D 29 to 71Group 1: GA201-ge, 1.5 mg/kg/occasion 623m 2.73 2.61 2.74 2.71 2.75+0.06 +0.03 (+0.02) (−0.12) (+0.13) (−0.03) (+0.04) 590f 3.16 3.11 3.183.27 3.23 +0.27 +0.17 (−0.03) (−0.05) (+0.07) (+0.09) (−0.04) Group 2:GA201-ge, 4.5 mg/kg/occasion 461m 2.45 2.54 2.60 2.59 +0.03 +0.06(−0.08) (+0.09) (+0.06) (−0.01) 462f 2.95 3.05 3.02 2.96 +0.05 +0.08(+0.04) (+0.10) (−0.03) (−0.06) Group 3: GA201-ge, 12 mg/kg/occasion463m 2.71 2.92 2.98 3.09 +0.23 +0.28 (−0.04) (+0.21) (+0.06) (+0.11)612f 2.81 3.04 3.05 3.10 +0.18 +0.09 (−0.10) (+0.23) (+0.01) (0.05)Conclusions

There was no effect of treatment at the injection sites and no clinicalfindings considered to be related to treatment with Glyco-mAb(anti-EGFR). Bodyweight changes were within normal expected ranges.There were no findings considered to be related to treatment atmacroscopic examination and organ weights of animals were within normalexpected ranges. In conclusion, treatment at 1.5, 4.5 or 12mg/kg/occasion was well tolerated with no clear findings of systemictoxicity.

EGFR is not a tumor specific target, since it is present on the surfaceof various normal tissues including liver, kidney and skin. Anti-EGFRantibodies with human IgG1 Fc region have previously been administeredto humans and have shown a tolerable side-effect profile (Vanhoefer, U.et al., Clin. Oncol. 2004 Jan. 1; 22(1):175-84; Needle M N, Semin Oncol.2002 October; 29 (5 Suppl 14):55-60). Clearly, there would besignificant concerns for administering to a human or other mammal ananti-EGFR antibody with significantly increased ADCC, due to enhancedkilling activity that could be displayed against critical normal tissuessuch as liver, kidney and skin. Surprisingly, the present inventors havefound that administering such an anti-EGFR antibody, Fc engineered asdescribed above and with up to 1000-fold increased ADCC activity, invivo to mammals did not lead to significant toxicities. Theconcentrations of antibody were kept above 1 microgram per milliliterfor at least 4 weeks (and above 100 micrograms per milliliter for someanimals). Such exposure levels are typical for antibody therapy. MaximalADCC for the antibody of this study is already achieved atconcentrations of 1 microgram per milliliter. Single doseadministrations of doses of 40 and 100 mg of anti-EGFR antibody (theparental rat ICR62 antibody) to human cancer patients have shownspecific targeting of tumors in vivo (Modjtahedi, H. et al., Br JCancer. 1996 January; 73(2):228-35.).

Cynomolgus monkey effector cells have highly-homologous FcgammaRIIIreceptor and have been shown to mediate enhanced ADCC with Fc engineeredantibodies (and with antibodies glycoengineered for increased levels ofnon-fucosylated oligosaccharides in the Fc region). The level of ADCCincrease is very similar to that observed with human effector cells(PBMCs).

In summary, we have found that anti-EGFR antibodies Fc engineered forincreased Fc-FcgammaRIII binding affinity and for increased ADCC can beadministered to mammals to give concentrations above 1 microgram ofantibody per milliliter of serum for a period of at least 4 weeks inorder to give drug exposures nornally associated with significantaccumulation of antibody on target cells in vivo, without leading tosignificant toxicity.

Toxicity of an antigen binding molecule of the present invention can bemeasured and/or determined using any of the methods and/or parameters(e.g. blood chemistry values, histopathological indicators, etc.)described herein above, or by any means known to those of skill in theart. A clinically significant level of toxicity is understood by one ofskill in the art to be a level that exceeds levels generally accepted bythe U.S. Food and Drug Administration for antibodies administeredclinically.

Example 5 Modifications to the Light Chain CDRs

Using methods described above, anti-EGFR light chain variable regionvariants were generated from the I-KC light chain variable regionconstruct (SEQ ID NO:43 and SEQ ID NO:45), wherein the sequence encodingthe amino acid residue at varous positions in the rat ICR62 CDRs werereplaced with the corresponding amino acid residue from a human germlinevariable gene sequence. Table 38 shows the substitutions that were madewithin the CDRs of the I-KC light chain variable region construct (SEQID NO:45): TABLE 38 Minimized Light Chain CDRs AMINO ACID LIGHT CHAINCDR SUBSTITUTION IN WHICH NAME OF MADE IN SUBSTITUTION WAS CONSTRUCT SEQID NO: 45 MADE I-KC1 N30R* CDR1 I-KC2 Y32W CDR1 I-KC3 N34G CDR1 I-KC4N50T CDR2 I-KC5 T51A CDR2 I-KC6 N52S CDR2 I-KC7 N53S CDR2 I-KC8 T56SCDR2 I-KC9 F94Y CDR3*Identified according to standard nomenclature (e.g., “N30R” means theAsparagine (N) residue at position 30 of SEQ ID NO: 45 is replaced withan argininen (R) residue).

All substitution residues identified above were derived from the humanVK1_(—)6 acceptor sequence except for the Y32W exchange, wherein the Wof a related human germline sequence was substituted for the Y atposition 32 in SEQ ID NO:45.

Each of the I-KC variant constructs (I-KC1 to I-KC9) was paired with aheavy chain variable region comprising construct I-HHD (SEQ ID NO:16 andSEQ ID NO:15) and a binding assay performed according to the methodsdescribed in the previous examples. Constructs I-KC1 to I-KC9 werecompared to the I-KC construct (SEQ ID NO:46 and SEQ ID NO:45) forbinding affinity to EGFR in A431 target cells (FIG. 29).

As seen in FIG. 29, only the modfication of residue 34 to itscorresponding human sequence (N34G) resulted in a slight decrease inbinding affinity (EC50 value increased by a factor of 10). All otherconstructs retained binding activity comparable to the I-KC construct(SEQ ID NO:45). Therefore, when paired with a chimeric (e.g., humanized)heavy chain construct specific for EGFR, the light chain can be entirelyhuman (e.g., from a human light chain V gene sequence) and still retainspecific binding for EGFR. In particular, CDR2 and CDR3 can be entirelyin human germline form.

Antigen Binding Molecules Comprising EGFR-Specific CDRs

The present invention therefore contemplates an antigen binding moleculecomprising a chimeric (e.g., humanized) heavy chain variable regioncomprising EGFR-specific CDRs paired with a light chain variable region,wherein the light chain variable region has fewer than ten non-humanamino acid residues. In other embodiments, the light chain variableregion has fewer than nine, eight seven, six, five, four, three, two, orone non-human amino acid residue(s). In preferred embodiments, the lightchain variable region has fewer than two or fewer than one (i.e., no)non-human amino acid residues. In one embodiment, the light chainvariable region comprises one or more human germline variable regiongene sequences. Human germline variable region gene sequences encodinglight chain variable regions are known in the art, and can be found, forexample, in the IMGT database, available athttp://imgt.cines.fr/home.html. In a preferred embodiment, the the humangermline sequence is derived from the VK1_(—)6 germline sequence. Inother embodiments, amino acid residues within the human germline lightchain variable region amino acid sequence can be substituted with one ormore residues from another human germline light chain variable regionsequence.

In one embodiment, the present invention is directed to an antigenbinding molecule comprising a sequence selected from the groupconsisting of SEQ ID NO.:1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ IDNo:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ IDNo:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID No:37; SEQ IDNo:39; and SEQ ID No:121, and a light chain comprising a polypeptideencoded by one or more human germline variable gene sequence. In apreferred embodiment, the human germline sequence is derived from theVK1_(—)6 germline sequence.

In another embodiment, the present invention is directed to an antigenbinding molecule comprising a sequence selected from the groupconsisting of: SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQ ID NO:124; (b) asequence selected from a group consisting of: SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, andSEQ ID NO:126; (c) SEQ ID NO:108; and (d) a polypeptide comprising ahuman light chain variable region encoded by one or more human germlinegene sequences. In a particular embodiment, the human germline sequenceis derived from the VK1_(—)6 germline sequence. In another embodiment,the human germline variable region gene sequence comprises the VK1_(—)6germline gene sequence with a substitution of one or more amino acidcodons with a sequence from a different human germline light chainvariable region gene sequence.

In other embodiments, the antigen binding molecule of the presentinvention comprises an EGFR-specific heavy chain variable region of thepresent invention, and a variant of SEQ ID NO:45. In one embodiment, thevariant of SEQ ID NO:45 comprises an amino acid substitution at one ormore positions in the complementarity determining regions (CDRs). Inspecific embodiments, the substitution is of an amino acid residue at aposition selected from the group consisting of: amino acid position 30of SEQ ID NO:45; amino acid position 32 of SEQ ID NO:45; amino acidposition 34 of SEQ ID NO:45; amino acid position 50 of SEQ ID NO:45;amino acid position 51 of SEQ ID NO:45; amino acid position 52 of SEQ IDNO:45; amino acid position 53 of SEQ ID NO:45; amino acid position 56 ofSEQ ID NO:45; amino acid position 94 of SEQ ID NO:45; and anycombination of substitutions thereof. In more specific embodiments, thesubstitution in SEQ ID NO:45 is selected from the group consisting of:substitution of an arginine (R) for the asparagine (N) at position 30 ofSEQ ID NO:45; substitution of a tryptophan (W) for the tyrosine (Y) atposition 32 of SEQ ID NO:45; substitution of a glycine (G) for theasparagine (N) at position 34 of SEQ ID NO:45; substitution of athreonine (T) for the asparagine (N) at position 50 of SEQ ID NO:45;substitution of an alanine (A) for the threonine (T) at position 51 ofSEQ ID NO:45; substitution of a serine (S) for the asparagine (N) atposition 52 of SEQ ID NO:45; substitution of a serine (S) for theasparagine (N) at position 53 of SEQ ID NO:45; substitution of a serine(S) for the threonine (T) at position 56 of SEQ ID NO:45; substitutionof a tyrosine (Y) for the phenylalanine (F) at position 94 of SEQ IDNO:45; and any combination thereof. In a particular embodiment, all ofthese substitutions of amino acid residues in SEQ ID NO:45 areincorporated in a single light chain variant. In preferred embodiments,antigen binding molecules comprising the light chain variants with aminoacid substitutions for the ICR62 CDRs retain specific binding to EGFR(as compared to an antigen binding molecule comprising a light chainvariable region comprising the sequence of SEQ ID NO:45) when the lightchain variant is paired with a polypeptide comprising a heavy chainvariable region of the present invention.

The present invention is also directed to polynucleotides that encodeany of the above polypeptides and/or antigen binding molecules. Thepresent invention is further directed to the antigen binding moleculesdescribed above, with a pharmaceutically acceptable carrier.

All publications such as textbooks, journal articles, GenBank or othersequence database entries, published applications, and patentapplications mentioned in this specification are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

1. An isolated polynucleotide comprising (a) a sequence selected fromthe group consisting of: SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQID NO:64, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:122, and SEQ ID NO:124; and (b) a sequence selected from the groupconsisting of: SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:96,SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, and SEQ IDNO:126; and (c) SEQ ID NO:108. 2-3. (canceled)
 4. An isolatedpolynucleotide comprising (a) SEQ ID NO:114; (b) a sequence selectedfrom the group consisting of SEQ ID NO:116 and SEQ ID NO:118; and (c)SEQ ID NO:119.
 5. An isolated polynucleotide according to claim 1 or 2,which encodes a fusion polypeptide. 6-23. (canceled)
 24. An isolatedpolynucleotide comprising a sequence having at least 80% identity to asequence selected from the group consisting of SEQ ID No:4; SEQ ID No:6;SEQ ID No:8; SEQ ID No:10; SEQ ID No:12; SEQ ID No:14; SEQ ID No:16; SEQID No:18; SEQ ID No:20; SEQ ID No:22; SEQ ID No:24; SEQ ID No:26; SEQ IDNo:28; SEQ ID No:30; SEQ ID No:32; SEQ ID No:34; SEQ ID No:36; SEQ IDNo:38; SEQ ID No:40, and SEQ ID NO:120.
 25. An isolated polynucleotidecomprising a sequence having at least 80% identity to a sequenceselected from the group consisting of SEQ ID No:46; SEQ ID No:50, SEQ IDNO:52. 26-38. (canceled)
 39. An isolated polynucleotide comprising asequence that encodes a polypeptide having a sequence selected from thegroup consisting of SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9;SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ ID No:19;SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ ID No:29;SEQ ID No:31; SEQ ID No:33; SEQ ID No:35; SEQ ID No:37; SEQ ID No:39;and SEQ ID NO:121.
 40. An isolated polynucleotide comprising a sequencethat encodes a polypeptide having a sequence selected from the groupconsisting of SEQ ID No:45, SEQ ID NO:49, and SEQ ID NO:51.
 41. Anexpression vector comprising an isolated polynucleotide according toclaim
 1. 42. (canceled)
 43. A host cell comprising the expression vectorof claim
 41. 44-46. (canceled)
 47. A polypeptide comprising a sequencederived from the rat ICR62 antibody and a sequence derived from aheterologous peptide.
 48. An antigen binding molecule comprising thepolypeptide of claim
 47. 49. An antigen binding molecule according toclaim 48, which binds human EGFR. 50-54. (canceled)
 55. A polypeptideaccording to claim 47, wherein said polypeptide comprises: (a) apolypeptide having a sequence selected from the group consisting of SEQID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:123, SEQ ID NO:59, SEQID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, and SEQ ID NO:125; (b) a polypeptide having asequence selected from the group consisting of SEQ ID NO:75 SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:127, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, and SEQ ID NO:105; and (c) a polypeptide havingthe sequence of SEQ ID NO:107.
 56. A polypeptide according to claim 47,wherein said polypeptide comprises: (a) a polypeptide having a sequenceof SEQ ID NO:111 or SEQ ID NO:113; (b) a polypeptide having a sequenceof SEQ ID NO:115; and (c) a polypeptide having a sequence of SEQ IDNO:117.
 57. A polypeptide according to claim 47, wherein saidpolypeptide comprises a sequence selected from the group consisting ofSEQ ID No: 1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ IDNo:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ ID No:19; SEQ IDNo:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ ID No:29; SEQ IDNo:31; SEQ ID No:33; SEQ ID No:35; SEQ ID No:37; SEQ ID No:39; and SEQID NO:121, or a variant thereof.
 58. A polypeptide according to claim47, wherein said polypeptide comprises a sequence selected from thegroup consisting of SEQ ID No: 43, SEQ ID No:45, SEQ ID NO:49, and SEQID NO:51, or a variant thereof.
 59. The isolated polypeptide accordingto claim 57 or 58, wherein said polypeptide is a fusion polypeptide. 60.(canceled)
 61. An antigen binding molecule comprising the isolatedpolypeptide of claim
 59. 62. The antigen binding molecule of claim 61,wherein said antigen binding molecule is an antibody. 63-67. (canceled)68. The antigen binding molecule of claim 61 wherein said antigenbinding molecule is an antibody fragment. 69-70. (canceled)
 71. Theantigen binding molecule of claim 61, wherein said antigen bindingmolecule is a recombinant antibody.
 72. The antigen binding molecule ofclaim 71, wherein said recombinant antibody is humanized.
 73. Theantigen binding molecule of claim 71, wherein said recombinant antibodycomprises a human Fc region.
 74. The antigen binding molecule of claim73, wherein said human Fc region is a human IgG Fc region.
 75. Anantigen binding molecule of claim 61, wherein said antigen bindingmolecule has been glycoengineered to have an altered oligosaccharidestructure in the Fc region.
 76. An antigen binding molecule according toclaim 75, wherein said Fc region has a reduced number of fucose residuesas compared to the nonglycoengineered antigen binding molecule.
 77. Anantigen binding molecule according to claim 75, wherein said Fc regionhas an increased proportion of bisected oligosaccharides as compared tothe nonglycoengineered antibody. 78-79. (canceled)
 80. An antigenbinding molecule according to claim 75, wherein said glycoengineeredantibody has an increased ratio of GlcNAc residues to fucose residues inthe Fc region compared to the nonglycoengineered antigen bindingmolecule. 81-86. (canceled)
 87. An antigen binding molecule according toclaim 75, wherein at least 50% of the oligosaccharides in the Fc regionare bisected. 88-91. (canceled)
 92. An antigen binding moleculeaccording to claim 75, wherein at least 50% of the oligosaccharides inthe Fc region are nonfucosylated. 93-95. (canceled)
 96. A method ofproducing an antigen binding molecule, which is capable of competingwith the rat ICR62 antibody for binding to human EGFR, and wherein saidantigen binding molecule is chimeric; said method comprising (a)culturing the host cell of claim 43 in a medium under conditionsallowing the expression of said polynucleotide encoding said antigenbinding molecule; and (b) recovering said antigen binding molecule. 97.The method of claim 96, wherein said antigen binding molecule is anantibody.
 98. The method of claim 96, wherein said chimeric antigenbinding molecule is humanized.
 99. A pharmaceutical compositioncomprising the antigen binding molecule according to claim 47 and apharmaceutically acceptable carrier.
 100. A method for treating anEGFR-related disorder comprising: (a) predicting a response to anti-EGFRtherapy in a human subject by assaying a sample from the human subjectbefore therapy with one or a plurality of reagents that detectexpression and/or activation of predictive biomarkers for cancer; (b)determining a pattern of expression and/or activation of one or more ofsaid predictive biomarkers, wherein the pattern predicts the humansubject's response to the anti-EGFR therapy; and (c) administering to ahuman subject who is predicted to respond the anti-EGFR directed therapya therapeutically effective amount of the composition of claim 99.101-105. (canceled)
 106. A method for targeting cells expressing EGFR ina subject comprising administering to said subject the pharmaceuticalcomposition of claim
 99. 107-109. (canceled)
 110. A method of treating acell proliferation disorder tractable by blocking EGFR-mediatedsignaling comprising administering a therapeutically effective amount ofthe pharmaceutical composition of claim 99 to a human subject in needthereof.
 111. A method for detecting in vivo or in vitro the presence ofEGFR in a sample comprising: (a) contacting a sample to be tested,optionally with a control sample, with the antigen binding molecule ofclaim 47 under conditions that allow for formation of a complex betweenthe antigen binding molecule and EGFR; and (b) detecting said antigenbinding molecule-EGFR complexes.
 112. A host cell engineered to expressat least one nucleic acid encoding a polypeptide havingβ(1,4)-N-acetylglucosaminyltransferase III activity in an amountsufficient to modify the oligosaccharides in the Fc region of apolypeptide produced by said host cell, wherein said polypeptide is anantigen binding molecule according to claim
 47. 113-145. (canceled) 146.An antigen binding molecule according to claim 73, wherein at least 50%of the oligosaccharides in the Fc region are nonfucosylated. 147-149.(canceled)
 150. The method according to claim 110, wherein said disorderis a cancer selected from the group consisting of breast cancer, bladdercancer, head & neck cancer, skin cancer, pancreatic cancer, lung cancer,ovarian cancer, colon cancer, prostate cancer, kidney cancer, and braincancer.
 151. An isolated polynucleotide comprising at least onecomplementarity determining region of the rat ICR62 antibody, or avariant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion, wherein said isolated polynucleotide encodes a fusionpolypeptide.
 152. An isolated polynucleotide according to claim 151,wherein said complementarity determining region is selected from thegroup consisting of: SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:126, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:14, SEQID NO:116, SEQ ID NO:118, and SEQ ID NO:119. 153-157. (canceled)
 158. Anisolated polynucleotide according to claim 151, wherein saidcomplementarity determining regions comprise at least one sequenceselected from the group consisting of: SEQ ID NO:54, SEQ ID NO:56, SEQID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQID NO:124; and at least one sequence selected from the group consistingof: SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:106, and SEQ ID NO:126; and SEQ ID NO:108, ortruncated forms of said sequences that contain at least thespecificity-determining residues for each of said complementaritydetermining regions. 159-164. (canceled)
 165. An isolated polypeptideencoded by the isolated polynucleotides of claim
 151. 166. An antigenbinding molecule according to claim 151, wherein said antigen bindingmolecule comprises at least, two, three, four, five, or sixcomplementarity determining region of the rat ICR62 antibody, or avariant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion(s), and further comprising a sequence derived from a heterologouspolypeptide. 167-169. (canceled)
 170. A method for producing an antigenbinding molecule having modified oligosaccharides in a host cell, saidmethod comprising: (a) culturing a host cell engineered to express atleast one nucleic acid encoding a polypeptide havingβ(1,4)-N-acetylglucosaminyltransferase III activity under conditionswhich permit the production of said antigen binding molecule, and whichpermit the modification of the oligosaccharides present on the Fc regionof said antigen binding molecule; and (b) isolating said antigen bindingmolecule wherein said antigen binding molecule is capable of competingwith the ICR62 antibody for binding to EGFR and wherein said antigenbinding molecule or fragment thereof is chimeric.
 171. A methodaccording to claim 170, wherein said modified oligosaccharides havereduced fucosylation as compared to non-modified oligosaccharides.172-179. (canceled)
 180. An antigen binding molecule engineered to haveincreased effector function produced by the method according to claim170.
 181. (canceled)
 182. An antigen binding molecule engineered to haveincreased Fc receptor binding affinity produced by the method of claim170.
 183. (canceled)
 184. An antigen binding molecule according to claim180, wherein said increased effector function is increased Fc-mediatedcellular cytotoxicity. 185-191. (canceled)
 192. An antigen bindingmolecule according to claim 182, wherein said Fc receptor is Fcactivating receptor.
 193. An antigen binding molecule according to claim192, wherein said Fc receptor is FcγRIIIa receptor.
 194. (canceled) 195.An antigen binding molecule produced by the method of claim 170, whereinsaid antigen binding molecule is a fusion protein that includes apolypeptide having a sequence selected from the group consisting of SEQID No:1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ IDNo:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ ID No:19; SEQ IDNo:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ ID No:29; SEQ IDNo:31; SEQ ID No:33; SEQ ID No:35; SEQ ID No:37; SEQ ID No:39, and SEQID NO:121, and a region equivalent to the Fc region of an immunoglobulinand engineered to have increased effector function.
 196. An antigenbinding molecule produced by the method of claim 170, wherein saidantigen binding molecule is a fusion protein that includes a polypeptidehaving a sequence selected from the group consisting of SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:49, and SEQ ID NO51, and a region equivalent tothe Fc region of an immunoglobulin and engineered to have increasedeffector function. 197-207. (canceled)
 208. A method of treating anEGFR-related disorder in a subject in need of such treatment comprisingadministering to said subject the antigen binding molecule of claim 48in an amount of from about 1.0 mg/kg to about 15 mg/kg. 209-216.(canceled)
 217. The method according to claim 208, wherein said disorderis a cancer selected from the group consisting of breast cancer, bladdercancer, head & neck cancer, skin cancer, pancreatic cancer, lung cancer,ovarian cancer, colon cancer, prostate cancer, kidney cancer, and braincancer. 218-238. (canceled)
 239. An antigen binding molecule accordingto claim 48, wherein said antigen binding molecule, when administered toa mammalian subject at concentrations above one microgram per milliliterof serum does not cause a clinically significant level of toxicity insaid mammalian subject. 240-241. (canceled)
 242. A method of treating anEGFR-related disorder in a mammal in need of treatment thereof, saidmethod comprising administering to said mammal an anitgen bindingmolecule according to claim 48, wherein said treatment results in serumconcentrations of said antigen binding molecule between about 1 andabout 100 μg/ml for a period of at least 4 weeks, and wherein saidtreatment does not cause a clinically significant level of toxicity insaid mammal. 243-244. (canceled)
 245. An isolated polynucleotideaccording to claim 39, further comprising a sequence encoding apolypeptide having a sequence selected from the group consisting of SEQID NO:43, SEQ ID NO:45, SEQ ID NO:59 and SEQ ID NO:51.